Luminescence-based methods and probes for measuring cytochrome P450 activity

ABSTRACT

The invention provides compounds, compositions, methods, substrates, and kits useful for analyzing the metabolic activity in cells, tissue, and animals and for screening test compounds for their effect on cytochrome P450 activity. In particular, a one-step and two-step methods using luminogenic molecules, e.g. luciferins or coelenterazines, that are cytochrome P450 substrates and that are also bioluminescent enzyme, e.g., luciferase, pro-substrates are provided. The present method further provides a method for stabilizing and prolonging the luminescent signal in a luciferase-based assay using luciferase stabilizing agents such as reversible luciferase inhibitors.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/665,314, filed Sep. 19, 2003, now U.S. Pat. No. 7,692,022 whichapplication claims the benefit of U.S. Provisional Application No.60/412,254, filed Sep. 20, 2002, and U.S. Provisional Application No.60/483,309, filed Jun. 27, 2003, each of which is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to methods, substrate compounds, and kitsfor analyzing the metabolic activity in animals, cells or in cell-freereaction formulations and for screening test compounds for their effecton metabolic activity. In particular, metabolic activity may be analyzedby employing a luminogenic molecule, e.g. luciferin derivative orcoelenterazines, as either a cytochrome P450 substrate or a dualcytochrome P450 substrate and bioluminescent enzyme pro-substrate. Inthe case where the luminogenic molecule is a cytochrome P450 substrateand a bioluminescent enzyme pro-substrate, P450 metabolism of theluminogenic molecule in a first reaction generates the substrate for abioluminescent enzyme. The bioluminescent enzyme then acts on thesubstrate in a second light-emitting reaction. P450 activity is thenascertained by measuring the luminescence of the reaction mixturerelative to a control reaction mixture. The present invention alsorelates to a method and kit for relieving inhibition of luciferase byits inhibitor inorganic pyrophosphate (iPP) using a pyrophosphatase suchas inorganic pyrophosphatase enzyme (iPPase).

BACKGROUND OF THE INVENTION

The presence and activity of enzymes can be used to determine the healthor metabolic state of a cell. Enzymes are also markers for the cell typesince the occurrence and activity of certain enzymes is frequentlycharacteristic of a particular cell. For instance, the activity ofcertain enzymes can often be used to distinguish cells of bacterial,plant or animal origin, or to distinguish the identity of tissue fromwhich the enzyme originates.

Detection of the presence and activity of enzymes can be facilitated bysubstrates that are converted by the enzyme of interest to a productthat has at least one property that can be measured. These reportermolecules include fluorescent and chromogenic substrates. Fluorescentsubstrates have been preferable because, in many cases, they have a veryhigh sensitivity and may permit measurements in living single cells withhigh spatial and temporal resolution. Chromogenic substrates can be veryspecific but often lack a high degree of resolution.

One family of enzymes useful for measuring the activity of living cellsor in extracts of cells is the Cytochrome P450 family. Cytochrome P450s(CYP450s) are a large family of heme-containing enzymes that, inaddition to the endogenous role in cell proliferation and development,includes many catalysts for detoxification and activation of lipophilicxenobiotics including therapeutic drugs, chemical carcinogens andenvironmental toxins. In some cases the metabolite(s) is more toxic thanthe parent compound. However, in other cases, metabolism of atherapeutic compound reduces the bioavailablity of the compound,lowering efficacy. This family of genes and the polymorphisms within thefamily play important roles in the interindividual variation in drugmetabolism, occurrence and severity of side effects and therapeuticfailures.

Hundreds of cytochrome P450s have been identified in diverse organismsincluding bacteria, fungi, plants, and animals (18). All CYP450s use aheme cofactor and share structural attributes. Most CYP450s are 400 to530 amino acids in length. The secondary structure of the enzyme isabout 70% alpha-helical and about 22% beta-sheet. The region around theheme-binding site in the C-terminal part of the protein is conservedamong cytochrome P450s. A ten amino acid signature sequence in this hemeiron ligand region has been identified which includes a conservedcysteine residue involved in binding the heme iron in the fifthcoordination site. In eukaryotic CYP450s, a membrane-spanning region isusually found in the first 15-20 amino acids of the protein, generallyconsisting of approximately 15 hydrophobic residues followed by apositively charged residue (18, 19.)

Some of the genes encoding CYP450s are inducible at the transcriptionlevel by the compounds they metabolize (1,2). The genes encoding CYP450shave been divided into families based on homology of deduced amino acidsequences (3). All mammals share at least 14 CYP450 families but mostdrug metabolism is catalyzed by only three families: CYP1, CYP2 andCYP3. Most of the P450 catalyzed drug metabolism in humans takes placein the liver and is accounted for by about 13 enzymes: CYP1A1, CYP1A2,CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1,CYP3A4, CYP3A5 and CYP3A7 (4).

Because of the central role CYP450s play in drug clearance, toxicity anddrug-drug interactions, CYP450s make useful targets for narrowing thefield of compounds that should be moved forward in the drug developmentprocess (5,8). Furthermore, knowledge of CYP450/drug interactions can bepredictive of drug disposition in a patient. There is a need forscreening assays that can be used in high throughput mode. Compoundswith properties that change in an easily detectable way upon oxidationby a CYP450 are useful as probes in high throughput assays for detectingeffects on CYP450 activity (6,7). There is also need for a method foranalyzing metabolic activity in cells under physiological conditions,using a substrate that is specific for CYP450 isozymes and yieldsproducts that are easily detectable. The signal should be detectable incell-free extracts of cells and in living cells and the assay shouldhave a low background signal.

Finally, there is a need to protect luciferase activity from itsinhibitor inorganic pyrophosphate. Although the inventors do not intendto limit the source of pyrophosphate, pyrophosphate may be present as acontaminant in orthophosphate salts used in buffers containing aluciferase-based reaction or may be generated as a product of aluciferase reaction with ATP, O₂ and luciferin.

SUMMARY OF THE INVENTION

Applicants have fulfilled these needs by providing methods, substratecompounds, and kits which can identify a compound, e.g., drug candidate,affecting a cytochrome P450 enzyme in a highly specific manner.

The invention provides luminogenic molecules that are useful as P450substrates or as dual P450 substrates and pro-substrates ofbioluminescent enzymes. In one embodiment of the invention, theluminogenic molecules are derivatives of(4S)-4,5-dihydro-2-(6-hydroxy-benzothiazolyl)-4-thiazolecarboxylic acid(D-luciferin) or2-(4-hydroxybenzyl)-6-(4-hydroxyphenyl)-8-benzyl-3,7-dihydroimidazo[1,2-a]pyrazine-3-one(coelenterazine) which are P450 substrates and pro-substrates ofluciferase. In the absence of prior P450 metabolism, these luciferinderivatives alone have limited or no capacity to interact withluciferase in light generating reactions. These compounds areselectively converted by CYP450s to light generating substrates forluciferase reactions and thereby provide the basis of assays with aluminescent readout.

The invention also provides a method for direct and indirectdetermination of P450 activity based on luminogenic molecules that arenatural coelenterazine and coelenterazine derivatives (collectivelyreferred to as coelenterazines).

The invention also provides methods for using luminogenic molecules todetermine whether a candidate drug (or class of candidate drugs) is aCYP450 enzyme substrate or regulator or CYP450 gene regulator, andrelated methods for selecting a candidate drug that will not be tooefficiently metabolized by at least one CYP450 enzyme and/or that thedrug will not act as an inhibitor of at least one CYP450 enzyme, and/orelicit an unfavorable drug-drug interaction. Methods of screening acandidate drug (or libraries of drug candidates) of the presentinvention may be performed by a one-step or a two-stepCYP450/bioluminescent enzyme method in a cell-free, cell-based,tissue-based or animal-based environment or may be part of a high orultra high throughput screening of libraries of drug candidates.

The invention also provides a method and kit for relieving inhibition ofluciferase by its inhibitor inorganic pyrophosphate (iPP) which may bepresent as a contaminant or generated as a product of a luciferase-basedreaction with ATP, O2 and luciferin.

The invention also provides a method and kit for enhancing orstabilizing a luminescent signal in a luciferase-based reaction thatemploys a reversible luciferase inhibitor.

Thus, in one embodiment of the invention, a method is provided formeasuring the activity of a cytochrome P450 enzyme comprising:

(a) providing a luminogenic molecule wherein the molecule is acytochrome P450 substrate and a pro-substrate of bioluminescent enzyme;

(b) contacting the luminogenic molecule with at least one cytochromeP450 enzyme and at least one bioluminescent enzyme to produce a reactionmixture; and

(c) determining cytochrome P450 activity by measuring luminescence ofthe reaction mixture.

In one aspect of this embodiment of the invention, step (b) furtherincludes a pyrophosphatase such as an inorganic pyrophosphatase.

In another aspect of this embodiment of the invention, the luminogenicmolecule, the cytochrome P450 enzyme, and the bioluminescent enzyme arecontacted at about the same time.

In another aspect of this embodiment of the invention, the luminogenicmolecule is contacted with at least one cytochrome P450 enzyme to form afirst reaction mixture prior to contacting with the bioluminescentenzyme to form a second reaction mixture.

In another aspect of this embodiment of the invention, the secondreaction mixture further comprises a detergent, preferably a non-ionicdetergent.

In another embodiment of the invention, a method is provided formeasuring cytochrome P450 enzyme activity in a cell comprising:

(a) providing a luminogenic molecule wherein the molecule is acytochrome P450 substrate and a pro-substrate of bioluminescent enzyme;

(b) contacting a cell with the luminogenic molecule and a bioluminescentenzyme to produce a mixture; and

(c) determining cytochrome P450 activity of the cell by measuringluminescence of the mixture.

In one aspect of this embodiment of the invention, cell is recombinantand expresses the bioluminescent enzyme.

In another aspect of this embodiment of the invention, step (b) cell isfurther contacted with a lysis reagent.

In another aspect of this embodiment of the invention, the cell is lysedprior to step (b).

In another aspect of this embodiment of the invention, the cell is lysedprior to step (c).

In another aspect of this embodiment of the invention, the cell iscontacted first with the luminogenic molecule to produce a firstreaction mixture prior to contact with the bioluminescent enzyme toproduce a second reaction mixture. The second reaction mixture mayfurther comprises a detergent such as a non-ionic detergent and/or apyrophosphatase such as an inorganic pyrophosphatase.

In another embodiment of the invent, a method is provided for measuringcytochrome P450 enzyme activity in animal tissue comprising:

(a) providing a luminogenic molecule wherein the molecule is acytochrome P450 substrate and a pro-substrate of bioluminescent enzyme;

(b) contacting an animal tissue with the luminogenic molecule and abioluminescent enzyme to provide a mixture; and

(c) determining cytochrome P450 activity of the tissue by measuringluminescence of the mixture.

In one aspect of this embodiment of the invention, the tissue iscontacted first with the luminogenic molecule for a first predeterminedtime period prior to contact with the bioluminescent enzyme to provide asecond mixture. The second reaction mixture may further comprise adetergent such as a non-ionic detergent and/or a pyrophosphatase such asinorganic pyrophosphatase.

In another embodiment of the invention, a method is provided formeasuring cytochrome P450 enzyme activity in an animal comprising:

(a) providing a luminogenic molecule wherein the molecule is acytochrome P450 substrate and a pro-substrate of bioluminescent enzyme;

(b) administering the luminogenic molecule to the animal;

(c) obtaining a biological sample from the animal; and

(d) contacting the biological sample with a bioluminescent enzyme toform a reaction mixture; and

(e) determining cytochrome P450 activity of the animal by measuringluminescence.

In one aspect of this embodiment of the invention, the reaction mixturefurther comprises a detergent such as a non-ionic detergent and/or apyrophosphatase such as an inorganic pyrophosphatase.

In another embodiment of the invention, a method is provided formeasuring cytochrome P450 enzyme activity in a transgeneic animal havinga bioluminescent enzyme transgene, said method comprising:

(a) providing a luminogenic molecule wherein the molecule is acytochrome P450 substrate and a pro-substrate of bioluminescent enzyme;

(b) administering the luminogenic molecule to a transgeneic animalhaving a bioluminescent enzyme transgene; and

(c) determining cytochrome P450 activity of the animal by measuringluminescence of tissue from the transgeneic animal.

In another aspect of this embodiment of the invention, thebioluminescent enzyme transgene is a luciferase transgene.

In another embodiment of the invention, a method is provided forscreening a compound for its effect on cytochrome P450 activitycomprising:

(a) providing a compound for screening;

(b) providing a luminogenic molecule wherein the molecule is acytochrome P450 substrate and a pro-substrate of bioluminescent enzyme;

(c) contacting the compound, the luminogenic molecule, at least onecytochrome P450 enzyme, and a bioluminescent enzyme to produce areaction mixture; and

(d) determining cytochrome P450 activity, if any, resulting from theinteraction of the compound with the cytochrome P450 enzyme by measuringluminescence of the reaction mixture.

In one aspect of this embodiment of the invention, the compound,luminogenic molecule, the cytochrome P450 enzyme, and the bioluminescentenzyme are contacted at about the same time.

In another aspect of this embodiment of the invention, the compound,luminogenic molecule and at least one cytochrome P450 enzyme arecontacted first to form a first reaction mixture prior to contactingwith the bioluminescent enzyme to form a second reaction mixture. Thesecond reaction mixture further includes a detergent such as a non-ionicdetergent and/or a pyrophosphatase such as an inorganic pyrophosphatase.

In another aspect of this embodiment of the invention, the compound iscontacted first with the one or more cytochrome P450 enzymes to form afirst reaction mixture, the first reaction mixture are then contactedwith the luminogenic molecule to form a second reaction mixture, and thesecond reaction mixture is then contacted with a bioluminescent enzymeto form a third reaction mixture. The third reaction mixture may furtherinclude a detergent such as a non-ionic detergent and/or apyrophosphatase such as an inorganic pyrophosphatase.

In another embodiment of the invention, a method is provided fordetermining the effect of a compound on cytochrome P450 enzyme activityof a cell comprising the steps of:

(a) providing a compound for testing (b) contacting a cell with a testcompound, a luminogenic molecule and a bioluminescent enzyme, whereinthe luminogenic molecule is a cytochrome P450 substrate and apro-substrate of bioluminescent enzyme; and

(c) determining cytochrome P450 enzyme activity of the cell, if any,resulting from the exposure of the cell to the test compound bymeasuring and comparing luminescence from said cell with a second cellnot exposed to the test compound.

In one aspect of this embodiment of the invention, the cell isrecombinant and expresses the bioluminescent enzyme.

In another aspect of this embodiment of the invention, the cell iscontacted first with the compound to produce a first reaction mixtureprior to contact with the luminogenic molecule to produce a secondreaction mixture. The second mixture may further comprise abioluminescent enzyme. The bioluminescent enzyme may be added to thesecond reaction mixture after a predetermined time period. The secondreaction mixture further includes a detergent such as a non-ionicdetergent and/or a pyrophosphate such inorganic pyrophosphate.

In another aspect of this embodiment of the invention, step (b) furtherincludes a pyrophosphatase such as an inorganic pyrophosphatase.

In another embodiment of the invention, a method is provided fordetermining the effect of a compound on cytochrome P450 enzyme activityof animal tissue comprising the steps of:

(a) providing a test compound;

(b) contacting an animal tissue with the test compound, a luminogenicmolecule and a bioluminescent enzyme, wherein the luminogenic moleculeis a cytochrome P450 substrate and a pro-substrate of bioluminescentenzyme; and

(c) determining cytochrome P450 enzyme activity of the tissue, if any,resulting from the exposure of the tissue to the test compound bymeasuring and comparing luminescence from said tissue with a controltissue not exposed to the test compound.

In one aspect of this embodiment of the invention, the animal tissueexpresses the bioluminescent enzyme.

In another aspect of this embodiment of the invention, the tissue iscontacted with the test compound to produce a first mixture prior tocontact with the luminogenic molecule to produce a second mixture. Thesecond mixture further comprises a bioluminescent enzyme. Thebioluminescent enzyme may added to the second reaction mixture after apredetermined time period. The second reaction mixture may furtherinclude a detergent such as a non-ionic detergent.

In another aspect of this embodiment of the invention, step (b) mayfurther include a pyrophosphatase such as an inorganic pyrophosphatase.

In another embodiment of the invention, a method is provided fordetermining the effect of a compound on cytochrome P450 enzyme activityin an animal comprising:

(a) providing a compound for testing;

(b) administering the test compound to an animal;

(c) administering a luminogenic molecule to the animal, wherein theluminogenic molecule is a cytochrome P450 substrate and a pro-substrateof bioluminescent enzyme;

(d) obtaining a biological sample from said animal;

(e) contacting the biological sample with a bioluminescent enzyme; and

(f) determining cytochrome P450 enzyme activity of said animal afterexposure of said animal to the test compound by measuring and comparingluminescence from said biological sample with a second biological sampletaken from an animal not exposed to said test compound.

In one aspect of this embodiment of the invention, step (c) is performedafter step (b) after a predetermined time period has elapsed.

In another aspect of this embodiment of the invention, the biologicalsample is taken from the animal just prior to exposure to the testcompound.

In another aspect of this embodiment of the invention, the biologicalsample comprises blood, serum, bile, urine, feces, or tissue.

In another embodiment of the invention, a method is provided fordetermining the effect of a compound on cytochrome P450 enzyme activityin an transgeneic animal having a bioluminescent enzyme transgene, saidmethod comprising:

(a) providing a compound for testing;

(b) administering the test compound to a transgeneic animal having abioluminescent enzyme transgene;

(c) administering a luminogenic molecule to the animal, wherein theluminogenic molecule is a cytochrome P450 substrate and a pro-substrateof the bioluminescent enzyme; and

(d) determining cytochrome P450 enzyme activity of said animal afterexposure of said animal to the test compound by measuring and comparingluminescence from tissue from said transgeneic animal with a secondbiological sample taken from another transgeneic animal not exposed tosaid test compound.

In one aspect of this embodiment of the invention, step (c) is performedafter step (b) after a predetermined time period has elapsed.

In another aspect of this embodiment of the invention, thebioluminescent enzyme transgene is a luciferase transgene.

In another embodiment of the invention, a high throughput method isprovided for rapidly screening a plurality of compounds to determinetheir effect on cytochrome P450 activity, said method comprising:

(a) providing compounds for screening;

(b) contacting the compounds to be screened with (i) a luminogenicmolecule wherein the luminogenic molecule is a cytochrome P450 substrateand a pro-substrate of bioluminescent enzyme; (ii) one or morecytochrome P450 enzymes; and (iii) one or more bioluminescent enzymes toform reaction mixtures, each reaction mixture having one or morecompounds; and

(c) determining cytochrome P450 enzyme activity, if any, resulting fromthe interaction of one or more compounds with one or more cytochromeP450 enzymes by measuring luminescence of the reaction mixtures.

In one aspect of this embodiment of the invention, the compounds arecontacted first with the one or more cytochrome P450 enzymes to formfirst reaction mixtures, the first reaction mixtures are then contactedwith the luminogenic molecule to form second reaction mixtures, and thesecond reaction mixtures are then contacted with a bioluminescent enzymeto form third reaction mixtures. The third reaction mixture may furtherinclude a detergent such as a non-ionic detergent.

In another aspect of this embodiment of the invention, the compounds arecontacted first with one or more cytochrome P450 enzymes and theluminogenic molecule to form first reaction mixtures prior to contactwith one or more bioluminescent enzymes to form a second reactionmixture. The second reaction mixture may further comprise a detergentsuch as a non-ionic detergent.

In another aspect of this embodiment of the invention, the compounds arecontacted simultaneously or contemporaneously with the one or morecytochrome P450 enzymes and the luminogenic molecule to form firstreaction mixtures prior to contacting with one or more bioluminescentenzymes to form second reaction mixtures.

In another aspect of this embodiment of the invention, step (b) furthercomprises a pyrophosphatase such as an inorganic pyrophosphatase.

In another embodiment of the invention, a high throughput method isprovided for rapidly screening a plurality of compounds to determinetheir effect on cytochrome P450 activity of a cell, said methodcomprising:

(a) providing compounds for screening;

(b) contacting cells with the compounds to be screened, a luminogenicmolecule, and one or more bioluminescent enzymes to form reactionmixtures, wherein the luminogenic molecule is a cytochrome P450substrate and a pro-substrate of bioluminescent enzyme and each reactionmixture having one or more compounds;

(c) determining cytochrome P450 enzyme activity, if any, resulting fromthe interaction of one or more compounds with one or more cytochromeP450 enzymes by measuring luminescence of the reaction mixtures.

In one aspect of this embodiment of the invention, the cells arerecombinant and express bioluminescent enzyme.

In another aspect of this embodiment of the invention, thebioluminescent enzyme from an exogenous source is used.

In another aspect of this embodiment of the invention, steps (b) and/or(c) further comprises a pyrophosphatase such as an inorganicpyrophosphatase.

In another aspect of this embodiment of the invention, the cells arefirst contacted with the compounds and luminogenic molecule for a firstpredetermined time period, then contacted with the bioluminescent enzymefor a second predetermined time period. Detergent such as non-ionicdetergent may be present during the second predetermined time period.

In another aspect of this embodiment of the invention, the cells arefirst contacted with the compounds for a first predetermined timeperiod, then contacted with the luminogenic molecule for a secondpredetermined time period, then contacted with the bioluminescent enzymefor a third predetermined time period. Detergent such as non-ionicdetergent may present in the mixture during the third predetermined timeperiod.

In another aspect of this embodiment of the invention, the cells arefirst contacted with the compounds for a first predetermined timeperiod, then contacted with the luminogenic molecule and bioluminescentenzyme for a second predetermined time period.

In another aspect of this embodiment of the invention, the cells,compounds, luminogenic molecule, and bioluminescent enzyme are contactedsimultaneously.

In another embodiment of the invention, a high throughput method isprovided for rapidly screening a plurality of compounds to determinetheir effect on cytochrome P450 activity of animal tissue, said methodcomprising:

(a) providing compounds for screening;

(b) contacting animal tissue with the compounds to be screened, aluminogenic molecule, and one or more bioluminescent enzymes to formreaction mixtures, wherein the luminogenic molecule is a cytochrome P450substrate and a pro-substrate of bioluminescent enzyme and each reactionmixture having one or more compounds;

(c) determining cytochrome P450 enzyme activity, if any, resulting fromthe interaction of one or more compounds with one or more cytochromeP450 enzymes by measuring luminescence of the reaction mixtures.

In one aspect of this embodiment of the invention, the tissue expressesat least one bioluminescent enzyme.

In another aspect of this embodiment of the invention, the tissue isfirst contacted with the compounds and luminogenic molecule for a firstpredetermined time period prior to contact with the bioluminescentenzyme. Detergent such as non-ionic detergent may be added after thefirst predetermined time period. Detergent and bioluminescent enzyme maybe added at the same time.

In another aspect of this embodiment of the invention, detergent isadded prior to addition of the bioluminescent enzyme.

In another aspect of this embodiment of the invention, the tissue isfirst contacted with the compounds for a first predetermined timeperiod, then contacted with the luminogenic molecule for a secondpredetermined time period, then contacted with the bioluminescent enzymefor a third predetermined time period. Detergent such as non-ionicdetergent may be added after the second predetermined time period.Detergent and bioluminescent enzyme may be added at the same time.Detergent and bioluminescent enzyme may be added at the same time.

In another aspect of this embodiment of the invention, the tissue isfirst contacted with the compounds for a first predetermined timeperiod, then contacted with the luminogenic molecule and bioluminescentenzyme for a second predetermined time period.

In another aspect of this embodiment of the invention, the tissue,compounds, luminogenic molecule, and bioluminescent enzyme are contactedsimultaneously. In another aspect of this embodiment of the invention,steps (b) or (c) further comprises (iv) a pyrophosphatase such as aninorganic pyrophosphatase.

In another embodiment of the invention, a high throughput method isprovided for rapidly screening a plurality of compounds to determinetheir effect on cytochrome P450 activity of animal, said methodcomprising:

(a) providing compounds for screening;

(b) contacting a living teleost with the compounds to be screened, aluminogenic molecule, and a bioluminescent enzyme to form reactionmixtures, wherein the luminogenic molecule is a cytochrome P450substrate and a pro-substrate of bioluminescent enzyme and each reactionmixture having one or more compounds;

(c) determining cytochrome P450 enzyme activity, if any, resulting fromthe interaction of one or more compounds with one or more cytochromeP450 enzymes by measuring luminescence of the reaction mixtures thatinclude test compounds in comparison to control mixtures without testcompounds.

In one aspect of this embodiment of the invention, the telost istransgeneic and expresses bioluminescent enzyme.

In another aspect of this embodiment of the invention, the telosts arefirst contacted with the compounds and luminogenic molecule for a firstpredetermined time period prior to contact with the bioluminescentenzyme.

In another aspect of this embodiment of the invention, the telosts arefirst contacted with the compounds for a first predetermined timeperiod, then contacted with the luminogenic molecule for a secondpredetermined time period, then contacted with the bioluminescent enzymefor a third predetermined time period.

In another aspect of this embodiment of the invention, the telosts arefirst contacted with the compounds for a first predetermined timeperiod, then contacted with the luminogenic molecule and bioluminescentenzyme for a second predetermined time period.

In another aspect of this embodiment of the invention, the telosts,compounds, luminogenic molecule, and bioluminescent enzyme are contactedsimultaneously.

In another aspect of this embodiment of the invention, steps (b) or (c)further comprises (iv) a pyrophosphatase such as an inorganicpyrophosphatase.

In another embodiment of the invention, in any of the above methods, theluminogenic molecule is a luciferin derivative and the bioluminescentenzyme is a luciferase. Preferably the luciferin derivative has aformula:

wherein

-   R₁ represents hydrogen, hydroxyl, amino, C₁₋₂₀ alkoxy, substituted    C₁₋₂₀ alkoxy, C₂₋₂₀ alkenyloxy, substituted C₂₋₂₀ alkenyloxy,    halogenated C₂₋₂₀ alkoxy, substituted halogenated C₂₋₂₀ alkoxy,    C₃₋₂₀ alkynyloxy, substituted C₃₋₂₀ alkynyloxy, C₃₋₂₀ cycloalkoxy,    substituted C₃₋₂₀ cycloalkoxy, C₃₋₂₀ cycloalkylamino, substituted    C₃₋₂₀ cycloalkylamino, C₁₋₂₀ alkylamino, substituted C₁₋₂₀    alkylamino, di C₁₋₂₀ alkylamino, substituted diC₁₋₂₀ alkylamino,    C₂₋₂₀ alkenylamino, substituted C₂₋₂₀ alkenylamino, di C₂₋₂₀    alkenylamino, substituted di C₂₋₂₀ alkenylamino, C₂₋₂₀ alkenyl C₁₋₂₀    alkylamino, substituted C₂₋₂₀ alkenyl C₁₋₂₀ alkylamino, C₃₋₂₀    alkynylamino, substituted C₃₋₂₀ alkynylamino, di C₃₋₂₀ alkynylamino,    substituted di alkylamino, C₃₋₂₀ alkynyl C₂₋₂₀alkenylamino, or    substituted C₃₋₂₀ alkynyl C₂₋₂₀alkenylamino;-   R₂ and R₃ independently represents C or N;-   R₄ and R₅ independently represents S, O, NR₉ wherein R₉ represents    hydrogen or C₁₋₂₀ alkyl, CR₉R₁₀ wherein R₉ and R₁₀ independently    represent H, C₁₋₂₀ alkyl, or fluorine;-   R₆ represents CH₂OH; COR₁₁ wherein R₁ represents H, OH, C₁₋₂₀    alkoxide, C₂₋₂₀ alkenyl, or NR₁₂R₁₃ wherein R₁₂ and R₁₃ are    independently H or C₁₋₂₀ alkyl; or —OM⁺ wherein M⁺ is an alkali    metal or a pharmaceutically acceptable salt; and-   R₇ represents H, C₁₋₄ alkyl, C₁₋₂₀ alkenyl, halogen, or C₁₋₆    alkoxide,-   with the proviso that R₁ is not OH or NH₂, R₇ is not H, R₆ is not    COR₁₁, R₁₁ is not OH, R₃ and R₂ are not both carbon, and R₄ and R₅    are not both S at the same time (luciferin and aminoluciferin).

In another embodiment of the invention, in any of the above methods, theluminogenic molecule comprises coelenterazine or coelenterazinederivatives and the bioluminescent enzyme is a luciferase. Preferablythe coelenterazine derivative has a formula:

wherein

-   R₁ is C₁₋₂₀ alkyl, branched C₃₋₂₀ alkyl, C₃₋₂₀ cycloalkyl, aralkyl,    C₁₋₂₀ alkyl substituted with C₁₋₂₀ alkoxy, hydroxy, halogen, C₁₋₂₀    alkylamino, or diC₁₋₂₀ alkylamino, aralkyl substituted with C₁₋₂₀    alkoxy, hydroxy, halogen, C₁₋₂₀ alkylamino, or diC₁₋₂₀ alkylamino;    and-   R₂, R₃, and R₄ are independently hydrogen, C₁₋₂₀ alkyl, C₃₋₂₀    cycloalkyl, branched C₃₋₂₀ alkyl, aryl, aralkyl, C₁₋₂₀ alkyl    substituted with C₁₋₂₀ alkoxy, hydroxy, halogen, C₁₋₂₀ alkylamino,    or diC₁₋₂₀ alkylamino, aralkyl substituted with C₁₋₂₀ alkoxy,    hydroxy, halogen, C₁₋₂₀ alkylamino, or diC₁₋₂₀ alkylamino, aryl    substituted with C₁₋₂₀ alkoxy, hydroxy, halogen, C₁₋₂₀ alkylamino,    or di C₁₋₂₀ alkylamino. Preferably R₄ is aryl or aryl substituted    with C₁₋₂₀ alkoxy, hydroxy, halogen, C₁₋₂₀ alkylamino, or C₁₋₂₀    dialkylamino.

In another embodiment of the invention, a kit is provided fordetermining the effect of a substance on cytochrome P450 enzyme activitycomprising:

(a) one or more luminogenic molecules wherein the molecule is acytochrome P450 enzyme substrate and a pro-substrate of luciferaseenzyme; and

(b) directions for using the kit.

In one aspect of this embodiment of the invention, the kit furthercomprises one or more bioluminescent enzymes such as a luciferase.Examples of luciferase include, without limitation, firefly lucifereaseor Renilla luciferase.

In another aspect of this embodiment of the invention, the kit furthercomprises ATP and magnesium ions.

In another aspect of this embodiment of the invention, the kit furthercomprises a detergent such as a non-ionic detergent.

In another aspect of this embodiment of the invention, the kit furthercomprising a pyrophosphatase such as an inorganic pyrophosphatase.

In another aspect of this embodiment of the invention, the luminogenicmolecule is a D-luciferin derivative that is a substrate of a cytochromeP450 enzyme and a pro-substrate of a luciferase enzyme. Preferably theluciferin derivative has a formula:

wherein

-   R₁ represents hydrogen, hydroxyl, amino, C₁₋₂₀ alkoxy, substituted    C₁₋₂₀ alkoxy, C₂₋₂₀ alkenyloxy, substituted C₂₋₂₀ alkenyloxy,    halogenated C₂₋₂₀ alkoxy, substituted halogenated C₂₋₂₀ alkoxy,    C₃₋₂₀ alkynyloxy, substituted C₃₋₂₀ alkynyloxy, C₃₋₂₀ cycloalkoxy,    substituted C₃₋₂₀ cycloalkoxy, C₃₋₂₀ cycloalkylamino, substituted    C₃₋₂₀ cycloalkylamino, C₁₋₂₀ alkylamino, substituted C₁₋₂₀    alkylamino, di C₁₋₂₀ alkylamino, substituted diC₁₋₂₀ alkylamino,    C₂₋₂₀ alkenylamino, substituted C₂₋₂₀ alkenylamino, di C₂₋₂₀    alkenylamino, substituted di C₂₋₂₀ alkenylamino, C₂₋₂₀ alkenyl C₁₋₂₀    alkylamino, substituted C₂₋₂₀ alkenyl C₁₋₂₀ alkylamino, C₃₋₂₀    alkynylamino, substituted C₃₋₂₀ alkynylamino, di C₃₋₂₀ alkynylamino,    substituted di C₃₋₂₀ alkynylamino, C₃₋₂₀ alkynyl C₁₋₂₀ alkylamino,    substituted C₃₋₂₀ alkynyl C₁₋₂₀ alkylamino, C₃₋₂₀ alkynyl    C₂₋₂₀alkenylamino, or substituted C₃₋₂₀ alkynyl C₂₋₂₀alkenylamino;-   R₂ and R₃ independently represents C or N;-   R₄ and R₅ independently represents S, O, NR₈ wherein R₈ represents    hydrogen or C₁₋₂₀ alkyl, CR₉R₁₀ wherein R₉ and R₁₀ independently    represent H, C₁₋₂₀ alkyl, or fluorine;-   R₆ represents CH₂OH; COR₁₁ wherein R₁₁ represents H, OH, C₁₋₂₀    alkoxide, C₂₋₂₀ alkenyl, or NR₁₂R₁₃ wherein R₁₂ and R₁₃ are    independently H or C₁₋₂₀ alkyl; or —OM⁺ wherein M⁺ is an alkali    metal or a pharmaceutically acceptable salt; and-   R₇ represents H, C₁₋₆ alkyl, C₁₋₂₀ alkenyl, halogen, or C₁₋₆    alkoxide,-   with the proviso that R₁ is not OH or NH₂, R₇ is not H, R₆ is not    COR₁₁, R₁₁ is not OH, R₃ and R₂ are not both carbon, and R₄ and R₅    are not both S at the same time (luciferin and aminoluciferin).

In another aspect of this embodiment of the invention, the luminogenicmolecule comprises coelenterazine or a coelenterazine derivative.Preferably the coelenterazine derivative has a formula:

wherein

-   R₁ is C₁₋₂₀ alkyl, branched C₃₋₂₀ alkyl, C₃₋₂₀ cycloalkyl, aralkyl,    C₁₋₂₀ alkyl substituted with C₁₋₂₀ alkoxy, hydroxy, halogen, C₁₋₂₀    alkylamino, or diC₁₋₂₀ alkylamino, aralkyl substituted with C₁₋₂₀    alkoxy, hydroxy, halogen, C₁₋₂₀ alkylamino, or diC₁₋₂₀ alkylamino;    and-   R₂, R₃, and R₄ are independently hydrogen, C₁₋₂₀ alkyl, C₃₋₂₀    cycloalkyl, branched C₃₋₂₀ alkyl, aryl, aralkyl, C₁₋₂₀ alkyl    substituted with C₁₋₂₀ alkoxy, hydroxy, halogen, C₁₋₂₀ alkylamino,    or diC₁₋₂₀ alkylamino, aralkyl substituted with C₁₋₂₀ alkoxy,    hydroxy, halogen, C₁₋₂₀ alkylamino, or diC₁₋₂₀ alkylamino, aryl    substituted with C₁₋₂₀ alkoxy, hydroxy, halogen, C₁₋₂₀ alkylamino,    or di C₁₋₂₀ alkylamino. Preferably R₄ is aryl or aryl substituted    with C₁₋₂₀ alkoxy, hydroxy, halogen, C₁₋₂₀ alkylamino, or C₁₋₂₀    dialkylamino.

In another aspect of this embodiment of the invention, the kit furthercomprises a reversible luciferase inhibitor. Preferably, the reversibleluciferase inhibitor is 2-(4-aminophenyl)-6-methylbenzothiazole (APMBT)or 2-amino-6-methylbenzothiazole (AMBT).

In another embodiment of the invention, a D-luciferin derivative isprovided that is a substrate of a cytochrome P450 enzyme and apro-substrate of luciferase enzyme.

In another embodiment of the invention, a composition is provided whichcomprises the D-luciferin derivative that is a substrate of a cytochromeP450 enzyme and a pro-substrate of luciferase enzyme.

In one aspect of this embodiment of the invention, the compositionfurther comprises a pyrophosphatase such as an inorganicpyrophosphatase.

In another embodiment of the invention, a D-luciferin derivative isprovided having the formula:

wherein

-   R₁ represents hydrogen, hydroxy, C₁₋₂₀ alkoxy or C₁₋₂₀ alkenyloxy    wherein the alkoxy and alkenyloxy are substituted with halogen,    hydroxy, amino, cyano, azido, heteroaryl or aryl substituted with    haloalkyl; or-   R₁ represents C₃₋₂₀ alkynyloxy; cycloalkoxy, cycloalkylamino, C₁₋₂₀    alkylamino, diC₁₋₂₀ alkylamino, C₂₋₂₀ alkenylamino, diC₂₋₂₀    alkenylamino, C₂₋₂₀ alkenyl C₁₋₂₀alkylamino, C₃₋₂₀ alkynylamino,    diC₃₋₂₀ alkynylamino, C₃₋₂₀ alkynyl C₁₋₂₀alkylamino, or C₃₋₂₀    alkynyl C₂₋₂₀alkenylamino, wherein each of the above groups are    optionally substituted with halogen, hydroxy, amino, cyano, azido,    heteroaryl or aryl substituted with haloalkyl;-   R₂ and R₃ independently represent C or N;-   R₄ and R₅ independently represent S; O; NR₉ wherein R₉ represents    hydrogen or C₁₋₂₀ alkyl; CR₉R₁₀ wherein R₉ and R₁₀ independently    represent H, C₁₋₂₀ alkyl or fluorine;-   R₆ represents CH₂OH; COR₁₁ wherein R₁₁ represents hydrogen, hydroxy,    C₂₋₂₀ alkenyl, or —OM⁺ wherein M⁺ is an alkali metal or a    pharmaceutically acceptable salt; and-   R₇ represents hydrogen, C₁₋₆ alkyl, C₂₋₂₀ alkenyl, halogen or C₁₋₆    alkoxide,    provided that-   when R₁ is hydroxy, R₇ is not hydrogen, R₁₁ is not hydroxy, R₂ and    R₃ are not both carbon, and R₄ and R₅ are not both S (luciferin);-   when R₁ is hydrogen, R₇ is not hydrogen, R₁₁ is not hydroxy, R₂ and    R₃ are not both carbon, and R₄ and R₅ are not both S    (dehydroluciferin); and-   when R₁ is hydroxy, R₇ is not hydrogen, R₆ is not CH₂OH, R₂ and R₃    are not both carbon, and R₄ and R₅ are not both S (luciferol).

In one aspect of this embodiment of the invention, the compound isselected from the group consisting of

-   -   luciferin 6′ 2-chloroethyl ether;    -   luciferin 6′ 4-picolinyl ether;    -   luciferin 6′ 4-trifluoromethylbenzyl ether;    -   luciferin 6′ 2-picolinyl ether; or    -   luciferin 6′ 3-picolinyl ether.

In another aspect of this embodiment of the invention, the compound isselected from the group consisting of

-   -   luciferin 6′ benzyl ether;    -   luciferin 6′ 4-trifluoromethylbenzyl ether;    -   luciferin 6′ phenylethyl ether;    -   luciferin 6′ geranyl ether; or    -   luciferin 6′ prenyl ether.

In another aspect of this embodiment of the invention, a composition isprovided which comprises the D-luciferin derivative. The composition mayfurther comprise a pyrophosphatase such as an inorganic pyrophosphatase.

In another embodiment of the invention, a method is provided formeasuring P450 enzyme activity comprising

(a) providing a coelentrazine or a coelenterazine derivative that is aP450 substrate and is chemiluminescent;

(b) contacting a coelentrazine or coelenterazine derivative with atleast one cytochrome P450 enzyme to form a reaction mixture; and

(c) determining cytochrome P450 activity by measuring chemoluminescenceof the reaction mixture.

In another embodiment of the invention, a method is provided formeasuring cytochrome P450 enzyme activity in a cell comprising:

(a) providing a coelentrazine or a coelenterazine derivative that is aP450 substrate and is chemiluminescent;

(b) contacting a cell with the coelenterazine or coelenterazinederivative to form a reaction mixture; and

(c) determining cytochrome P450 activity of the cell by measuring thechemiluminescence of the reaction mixture.

In one aspect of this embodiment of the invention, step (b) cell isfurther contacted with a lysis agent.

In another aspect of this embodiment of the invention, the cell is lysedprior to step (b).

In another aspect of this embodiment of the invention, the cell is lyzedprior to step (c).

In another embodiment of the invention, a method is provided formeasuring cytochrome P450 enzyme activity in animal tissue comprising:

(a) providing coelenterazine or a coelenterazine derivative that is aP450 substrate and is chemiluminescent;

(b) contacting an animal tissue with the coelenterazine or acoelenterazine derivative and a bioluminescent enzyme to provide amixture; and

(c) determining cytochrome P450 activity of the tissue by measuringluminescence of the mixture.

In another embodiment of the invention, a method is provided formeasuring cytochrome P450 enzyme activity in an animal comprising:

(a) providing coelenterazine or a coelenterazine derivative that is aP450 substrate and is chemiluminescent;

(b) administering the coelentrazine or a coelenterazine derivative to ananimal;

(c) obtaining a biological sample from the animal; and

(d) determining cytochrome P450 activity of the animal by measuringchemiluminescence of the sample.

In another embodiment of the invention, a method is provided forscreening a compound for its effect on cytochrome P450 activitycomprising:

(a) providing a compound for screening;

(b) providing coelenterazine or a coelenterazine derivative that is asubstrate of cytochrome P450 and is chemiluminescent;

(c) contacting the compound, coelentrazine or a coelenterazinederivative, and a cytochrome P450 enzyme to produce a reaction mixture;and

(d) determining cytochrome P450 activity, if any, resulting from theinteraction of the compound with the cytochrome P450 enzyme by measuringchemiluminescence of the reaction mixture.

In another embodiment of the invention, a method is provided fordetermining the effect of a compound on cytochrome P450 enzyme activityof a cell comprising the steps of:

(a) providing a compound for testing;

(b) contacting a cell with a test compound and coelentrazine or acoelenterazine derivative that is a substrate of cytochrome P450 andthat is chemiluminescent; and

(c) determining cytochrome P450 enzyme activity of the cell, if any,resulting from the exposure of the cell to the test compound bymeasuring and comparing chemiluminescence from said cell with a secondcell not exposed to the test compound.

In another embodiment of the invention, a method is provided fordetermining the effect of a compound on cytochrome P450 enzyme activityof animal tissue comprising the steps of

(a) providing a test compound;

(b) contacting an animal tissue with a test compound and coelentrazineor a coelenterazine derivative that is a substrate of cytochrome P450and that is chemiluminescent; and

(c) determining cytochrome P450 enzyme activity of the tissue, if any,resulting from the exposure of the tissue to the test compound bymeasuring and comparing chemiluminescence from said tissue with acontrol tissue not exposed to the test compound.

In another embodiment of the invention, a method is provided fordetermining the effect of a compound on cytochrome P450 enzyme activityin an animal comprising:

(a) providing a compound for testing;

(b) administering the test compound to an animal;

(c) administering coelentrazine or a coelenterazine derivative that is asubstrate of cytochrome P450 and that is chemiluminescent;

(d) obtaining a biological sample from said animal;

(e) determining cytochrome P450 enzyme activity of said animal afterexposure of said animal to the test compound by measuring and comparingchemiluminescence from said biological sample with a second biologicalsample taken from an animal not exposed to said test compound.

In another embodiment of the invention, a high throughput method isprovided for rapidly screening a plurality of compounds to determinetheir effect on cytochrome P450 activity, said method comprising:

(a) providing compounds for screening;

(b) contacting the compounds to be screened with (i) coelentrazine or acoelenterazine derivative that is a substrate of cytochrome P450 andthat is chemiluminescent; and (ii) one or more cytochrome P450 enzymes,each reaction mixture having one or more compounds; and

(c) determining cytochrome P450 enzyme activity, if any, resulting fromthe interaction of one or more compounds with one or more cytochromeP450 enzymes by measuring chemiluminescence of the reaction mixtures.

In another embodiment of the invention, a high throughput method forrapidly screening a plurality of compounds to determine their effect oncytochrome P450 activity of a cell, said method comprising:

(a) providing compounds for screening;

(b) contacting cells with the compounds to be screened andcoelenterazine or a coelenterazine derivative that is a substrate ofcytochrome P450 and that is chemiluminescent to form reaction mixtures,each reaction mixture having one or more compounds;

(c) determining cytochrome P450 enzyme activity, if any, resulting fromthe interaction of one or more compounds with one or more cytochromeP450 enzymes by measuring chemiluminescence of the reaction mixtures.

In another embodiment of the invention, a high throughput method isprovided for rapidly screening a plurality of compounds to determinetheir effect on cytochrome P450 activity of animal tissue, said methodcomprising: (a) providing an animal tissue with CYP450 activity

(a) providing compounds for screening;

(b) contacting animal tissue with the compounds to be screened andcoelenterazine or a coelenterazine derivative that is a substrate ofcytochrome P450 and that is chemiluminescent to form reaction mixtures,each reaction mixture having one or more compounds;

(c) determining cytochrome P450 enzyme activity, if any, resulting fromthe interaction of one or more compounds with one or more cytochromeP450 enzymes by measuring chemiluminescence of the reaction mixtures.

In another embodiment of the invention, a high throughput method isprovided for rapidly screening a plurality of compounds to determinetheir effect on cytochrome P450 activity of animal, said methodcomprising:

(a) providing compounds for screening;

(b) contacting a living teleost with the compounds to be screened andcoelenterazine or a coelenterazine derivative that is a substrate ofcytochrome P450 and that is chemiluminescent to form reaction mixtures,each reaction mixture having one or more compounds;

(c) determining cytochrome P450 enzyme activity, if any, resulting fromthe interaction of one or more compounds with one or more cytochromeP450 enzymes by measuring chemiluminescence of the reaction mixturesthat include test compounds in comparison to control mixtures withouttest compounds.

In another embodiment of the invention, in any of the above methodsinvolving coleentrazine or derivative that is a substrate of cytochromeP450 and that is chemiluminescent, preferably the coelenterazinederivative has a formula:

wherein

-   R₁ is C₁₋₂₀ alkyl, branched C₃₋₂₀ alkyl, C₃₋₂₀ cycloalkyl, aralkyl,    C₁₋₂₀ alkyl substituted with C₁₋₂₀ alkoxy, hydroxy, halogen, C₁₋₂₀    alkylamino, or diC₁₋₂₀ alkylamino, aralkyl substituted with C₁₋₂₀    alkoxy, hydroxy, halogen, C₁₋₂₀ alkylamino, or diC₁₋₂₀ alkylamino;    and-   R₂, R₃, and R₄ are independently hydrogen, C₁₋₂₀ alkyl, C₃₋₂₀    cycloalkyl, branched C₃₋₂₀ alkyl, aryl, aralkyl, C₁₋₂₀ alkyl    substituted with C₁₋₂₀ alkoxy, hydroxy, halogen, C₁₋₂₀ alkylamino,    or diC₁₋₂₀ alkylamino, aralkyl substituted with C₁₋₂₀ alkoxy,    hydroxy, halogen, C₁₋₂₀ alkylamino, or diC₁₋₂₀ alkylamino, aryl    substituted with C₁₋₂₀ alkoxy, hydroxy, halogen, C₁₋₂₀ alkylamino,    or di C₁₋₂₀ alkylamino.

In one aspect of this embodiment of the invention, preferably R₄ is arylor aryl substituted with C₁₋₂₀ alkoxy, hydroxy, halogen, C₁₋₂₀alkylamino, or C₁₋₂₀ dialkylamino.

In another aspect of this embodiment of the invention, the coelentrazinederivative is coelenterazine HH, methoxycoelenterazine HH orcoelenterazine.

In another embodiment of the invention, a method is provided forenhancing the stability of a luminescent signal generated by aluciferase-based reaction mixture comprising contacting a luciferasewith a reversible luciferase inhibitor in an amount effective to enhancethe stability and prolong the lifetime of the luminescent signalrelative to the luminescent signal generated in a comparableluciferase-based reaction mixture in the absence of the inhibitor.

In one aspect of this embodiment of the invention, the reversibleluciferase inhibitor is a competitive inhibitor.

In another aspect of this embodiment of the invention, the reversibleluciferase inhibitor comprises 2-(4-aminophenyl)-6-methylbenzothiazole(APMBT) or 2-amino-6-methylbenzothiazole (AMBT).

In another aspect of this embodiment of the invention, the effectiveamount of the inhibitor ranges from about 1 micromolar to about 1millimolar in the reaction mixture.

In another aspect of this embodiment of the invention, the effectiveamount of the inhibitor ranges from about 1 micromolar and about 500micromolar in the reaction mixture.

In another aspect of this embodiment of the invention, the effectiveamount of the inhibitor ranges from about 10 micromolar to about 200micromolar in the reaction mixture.

In another aspect of this embodiment of the invention, the effectiveamount of the inhibitor ranges is about 100 micromolar in the reactionmixture.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Luminescent CYP450 reaction scheme.

FIG. 2. Structures: D-luciferin((4S)-4,5-dihydro-2-(6-hydroxy-benzothiazolyl)-4-thiazolecarboxylicacid) and D-luciferin derivatives.

FIG. 3. Two-step luminescent CYP450 reactions. D-luciferin derivativeswere incubated in a CYP450 reaction mix for 60 minutes at 37° C. beforecombining with a luciferase reaction mixture. In —CYP450 controls,CYP450 Sf9 cell microsomes were replaced with control (no CYP450) Sf9cell membranes (panels B, C, D and E) or H₂O (panel A) or both (panel F,G, H, and I). Luminescence was read within 12 minutes of combining thereactions on a Turner Reporter (panels A, B, C and E) or Berthold Orion(panels D, F, G, and H) luminometer.

FIG. 4. Time-dependence of CYP450/substrate incubation in two-stepluminescent CYP450 reactions. D-luciferin derivatives were incubated ina CYP450 reaction mix for the indicated times at 37° C. before combiningwith a luciferase reaction mixture. For —CYP450 controls CYP450 Sf9 cellmicrosomes were replaced with H₂O. Luminescence was read within 12minutes of combining the reactions on a Turner Reporter (panels A and C)or Berthold Orion (panel B) luminometer.

FIG. 5. Time course of light output from two-step luminescent CYP450reactions. Luc ME was incubated in CYP450 reaction mixes for 60 minutesat 37° C. before combining with a luciferase reaction mixture. In CYP450controls CYP450 Sf9 cell microsomes were replaced with H₂O. Luminescencewas read on a Turner Reporter luminometer beginning 3 minutes aftercombining the reactions and at successive intervals as indicated for 284minutes.

FIG. 6. One-step luminescent CYP450 assays at room temperature. Luc MEwas incubated in combined CYP450 and luciferase reaction mixes at roomtemperature (˜22° C.). For —CYP450 controls CYP450 Sf9 cell microsomeswere replaced with H₂O. CYP450 and a luciferase reaction mix were addedsimultaneously to a CYP450 reaction mix and light output was readimmediately (time=0). Readings were then taken every 4.25 minutes for15.5 hours on a Turner Reporter luminometer.

FIG. 7. One-step luminescent CYP450 assays at 37° C. D-luciferinderivatives were incubated in combined CYP450 and luciferase reactionmixes at 37° C. For —CYP450 controls CYP450 Sf9 cell microsomes werereplaced with H₂O. CYP450 and a luciferase reaction mix were addedsimultaneously to a CYP450 reaction mix and light output was readimmediately (time=0). Readings were then taken every 10 minutes for 3hours on a Turner 20/20 luminometer.

FIG. 8. Pooled human liver microsomes in two-step luminescent CYP450reactions. D-luciferin derivatives were incubated in a CYP450 reactionmix with pooled human liver microsomes for 60 minutes at 37° C. beforecombining with a luciferase reaction mixture. For controls livermicrosomes were replaced with control (no CYP450) Sf9 cell membranes.Luminescence was read within 12 minutes of combining the reactions on aBerthold Orion luminometer. Vehicle for sulfaphenazole, ketoconazole andalpha-naphthoflavone was 1% acetonitrile and 1 mg/mL bovine serumalbumin in H₂O. Values labeled “nt” are vehicle controls. Concentrationsof sulfaphenazole, ketoconazole and alpha-naphthoflavone in thereactions were 100 micromolar, 100 micromolar and 10 micromolar,respectively.

FIG. 9. Two-step detection of CYP450 de-picolinylase activity.D-luciferin derivatives luc2PE, luc3PE, and luc4PE were incubated in aCYP450 reaction mix for 60 minutes at 37° C. before combining with aluciferase reaction mixture. In this Figure, the bars labeled “Sf9” arethe controls. These are Sf9 cell membranes without CYP450 expression.Luminescence was read within 12 minutes of combining the reactions on aBerthold Orion luminometer.

FIG. 10. CYP450-catalyzed conversion of luciferin derivatives toluciferin. Luciferin derivatives (100 micromolar) were incubated in aCYP450 reaction mix for the indicated time intervals. At the end of eachtime interval, the reaction mixture was quenched with Tergitol to 0.1%(v/v), then frozen in liquid nitrogen. 95 microliter aliquots of thereaction mixture were analyzed by HPLC and luciferin was detected byfluorescence with excitation at 330 nm and emission at 520 nm. The zerotime points represent the luciferin content of the derivatives fromcontrols (no enzyme).

FIG. 11. Detection of Cyp450 inhibition by known CYP450 substrates.Luciferin derivatives as substrates for luminescent CYP450 assays wereevaluated as probes for detecting other CYP450 substrates. CYP450substrates tested were diclofenac for CYP2C9 and phenacetin for CYP1A1and CYP1A2. The reactions were performed as described in Example 1except the first step (CYP450 reaction) was in a 50 microliter reactionvolume with 1 picomole of CYP450. In the second step a 50 microliterluciferase reaction was added to give final concentrations of 50micrograms/mL Ultra Glo luciferase, 200 micromolar ATP, 0.1% Tergitol(v/v), 4.0 mM MgSO₄ and 100 mM Tricine pH 8.4. Panel A illustratesinhibition of CYP1A2 by phenacetin using Luc ME as substrate. Panel Billustrates inhibition of CYP1A1 by phenacetin using Luc CEE assubstrate. Panel C illustrates inhibition of CYP2C9 by diclofenac usingHLuc as substrate.

FIG. 12: P450 action on methoxy-coelenterazine HH, coelenterazine HH andcoelenterazine by chemiluminescent and bioluminescent detection. Panel Ashows bioluminescence from methoxy-coelenterazine-HH in relative lightunits (RLU) generated in a Renilla luciferase containing reactionfollowing incubation of methoxy-coelenterazine HH with (+) or without(−) various P450 isozymes. Panel B shows the fold increase inbioluminescence from reactions containing methoxy-coelenterazine HH andP450 (+P450 RLU/−P450 RLU). Panel C shows chemiluminescence frommethoxy-coelenterazine HH in RLU generated following incubation ofmethoxy-coelenterazine HH with (+) or without (−) various P450 isozymes.Panel D shows the fold increase in chemiluminescence from reactionscontaining methoxy-coelenterazine HH and P450 (+P450 RLU/−P450 RLU).Panel E shows bioluminescence from coelenterazine HH in RLU generated ina Renilla luciferase containing reaction following incubation ofcoelenterazine HH with (+) or without (−) various P450 isozymes. Panel Fshows the fold decrease in bioluminescence from reactions containingcoelenterazine HH and P450 (+P450 RLU/−P450 RLU). Panel G showschemiluminescence from coelenterazine HH in RLU generated followingincubation of coelenterazine HH with (+) or without (−) various P450isozymes. Panel H shows the decrease in chemiluminescence from reactionscontaining coelenterazine HH and P450 (+P450 RLU/−P450 RLU). Panel Ishows bioluminescence from coelenterazine in RLU generated in a Renillaluciferase containing reaction following incubation of coelenterazinewith (+) or without (−) various P450 isozymes. Panel J shows thedecrease in bioluminescence from reactions containing coelenterazine andP450 (+P450 RLU/−P450 RLU). Panel K shows chemiluminescence fromcoelenterazine in RLU generated following incubation of coelenterazinewith (+) or without (−) various P450 isozymes. Panel L shows thedecrease in chemiluminescence from reactions containing coelenterazineand P450 (+P450 RLU/−P450 RLU).

FIG. 13. Protection of luciferase from inhibitory buffer using yeastiPPase. Yeast inorganic pyrophosphatase was found to be effective inreversing iPP inhibition of luciferase when inhibitory KPO₄ buffer used.

FIG. 14. Inorganic pyrophosphatases protect luciferase frompyrophosphatase contamination. Inorganic pyrophosphatases from differentsources were found to reverse iPP inhibition of luciferase wheninhibitory KPO₄ buffer is used.

FIG. 15. Protection of luciferase from added iPP using iPPase. Inorganicpyrophosphatase was found to be effective in reversing iPP inhibition ofa luciferase-based reaction when iPP is added to the reaction.

FIG. 16. Cell-based Luminescent CYP450 Assays. Primary rat hepatocyteswere treated for 2 days with inducers of CYP450 gene expression: 5micromolar 3-methylcholanthrene (MC), 50 micromolar dexamthasone (Dex)or 50 micromolar rifampicin (Rif) and their vehicle controls, 0.05, 0.1and 0.1% DMSO, respectively (uninduced); and an inhibitor of CYP450: 100micromolar troleandomycin (Tro). The induction medium was then replacedwith 300 microliters of 100 micromolar luciferin-CEE (panels A and B),200 micromolar luciferin-BE or 200 micromolar luciferin-BE plus Trodissolved in hepatocyte culture medium and allowed to incubate for 4hours. 100 microliters of medium was then removed from wells andcombined with luciferin detection reagent (see example 15) and 200microliters of luciferin detection reagent was added to the remaining200 microliters of medium on cells. Luminescence from 200 microliters ofculture medium reactions (panels A & C) and cell lysate reactions(panels B & D) was quantified.

FIG. 17. Stabilization of luminescent signals using luciferaseinhibitors. Inhibition of luciferase by an inhibitor2-(4-aminophenyl)-6-methylbenzothiazole (APMBT) or 2-amino-6-methylbenzothiazole (AMBT) stabilizes the luminescent signal in a luminescentCYP450 assay. 50 microliter CYP1A1 reactions (0.5 μmol recombinantCYP1A1 enzyme, 30 μM Luciferin chloroethyl ether, 100 mM KPO₄, 1.3 mMNADP⁺, 3.3 mM glucose-6-phosphate, 3.3 mM MgCl₂, 0.02 unitglucose-6-phosphate dehydrogenase) were incubated at 37° C. for 20 min.After the incubation, 50 microliters of a luciferin detection reagent(100 micrograms/mL thermostable luciferase (from Photurispennsylvanica), 400 micromolar ATP, 0.6% Prionex, 2 units/mL iPPase, 200mM Tricine pH 8.4, 20 mM MgSO₄, 2% Tergitol) containing either 100micromolar APMBT, 100 micromolar AMBT, or no inhibitor were added toeach aliquot of CYP1A1 reaction. Luminescence was read immediately andat subsequent 5 minute intervals for 1 hour.

DETAILED DESCRIPTION OF THE INVENTION

As defined herein, the term “cytochrome P450” or “CYP450” or “P450enzyme,” unless specified otherwise, refers to a family of heme-basedoxidase enzymes involved in the metabolism of hydrophobic drugs,carcinogens, and other potentially toxic compounds and metabolitescirculating in blood. It is known that the liver is the major organ forxenobiotic metabolism, containing high levels of the most importantCYP450 mixed-function oxidases. These mixed-function oxidases aredivided into subfamilies, which include CYP1A, 2A, 2C, 2D, 2E, and 3A.Within these subfamilies, there are numerous human P450 enzymes, oftentermed “isozymes” or “isoforms.” The human CYP3A, CYP2D6, CYP2C, andCYP1A isoforms are known to be important in drug metabolism. See, e.g.,Murray, M., 23 Clin. Pharmacokinetics 132-46 (1992). CYP3A4 is by farthe major isoform in human liver and the small intestines, comprising30% and 70% respectively of the total CYP450 protein in those tissues.Based primarily on in vitro studies, it has been estimated that themetabolism of 40% to 50% of all drugs used in humans involve CYP3A4catalyzed oxidations. See Thummel, K. E. & Wilkinson, G. R., In Vitroand In Vivo Drug Interactions Involving Human CYP 3A, 38 Ann. Rev.Pharmacol. Toxicol., 389-430 (1998).

The term “luminescent”, as used herein, includes bio-luminescence (i.e.light produced by a living organism), or chemi-luminescence (lightproduced when a chemical reaction proceeds). When the enzyme involvedhas evolved in an organism by natural selection for the purpose ofgenerating light, or the enzyme involved is a mutated derivative of suchan enzyme, the luminescent reactions are also called “bioluminescentreactions” and the enzyme involved is also called a “bioluminescentenzyme.” Examples of bioluminescent enzymes include, without limitation,firefly luciferase, Renilla luciferase, Cypridina luciferase, Aequorinphotoprotein, Obelin photoprotein, and the like.

The term “luminogenic molecule” as used herein refers to a moleculecapable of creating light via a chemical or biochemical reaction (e.g.beetle luciferin (or D-luciferin), coelenterazine, or a functionalanalog thereof). The luminogenic molecule could be either a P450substrate or a P450 substrate/bioluminescent enzyme pro-substrate.Generally, a luminogenic molecule is either a high-energy molecularspecies (e.g. a stabilized dioxetane), or it is transformed into ahigh-energy molecular species by a chemical reaction. The chemicalreaction is usually oxidation by oxygen, superoxide, or peroxide. Ineach case, the energy within the luminogenic molecule is released by thechemical reaction. Although at least some of this energy is released asphotons of light, the energy can also be released in other forms, suchas heat. The luminogenic molecules that do not yield light dispersetheir energy through alternative modes, often termed “dark pathways”.

The term “luciferin derivative” as used herein refers to a type ofluminogenic molecule or compound having a substantial structure ofD-luciferin and maybe a substrate of one or more cytochrome P450 enzymesand a pro-substrate of luciferase. In the presence of cytochrome P450,the compound is metabolized into luciferin, a substrate of luciferase.In the absence of prior P450 metabolism, some of the compound(s) maybind to luciferase as evidenced by their capacity to inhibit a reactionwith luciferin (data not shown), however, they are not turned over assubstrate in light-generating reactions. Without being bound by anytheory of operation, it is believed that these compounds are most likelycompetitive inhibitors of luciferase.

The term “coelenterazines” as used herein refers to naturalcoelenterazines and coelenterazine derivatives. Coelenterazines areknown to luminesce when acted upon by a wide variety of bioluminescentproteins, specifically marine luciferases. Examples of marineluciferases include Renilla luciferase, aequorin, Gaussia luciferase,Oplophorus luciferase, and Cypridina luciferase. Useful, butnon-limiting, coelenterazines are disclosed in U.S. patent applicationSer. No. 10/053,482, filed Nov. 2, 2001, the disclosure which is herebyincorporated by reference in its entirety. Coelenterazines are availablefrom Promega Corporation, Madison, Wis. and from Molecular Probes, Inc.,Eugene, Oreg. Coelenterazines may also be synthesized as described forexample in Shimomura et al., Biochem. J. 261: 913-20, 1989; Inouye etal., Biochem. Biophys. Res. Comm. 233: 349-53, 1997; and Teranishi etal., Anal. Biochem. 249: 37-43, 1997.

The term “luciferase,” unless specified otherwise, refers to a naturallyoccurring or mutant luciferase. The luciferase, if naturally occurring,may be obtained easily by the skilled from an organism. If theluciferase is one that occurs naturally or is a mutant, which retainsactivity in the luciferase-luciferin reaction, of a naturally occurringluciferase, it can be obtained readily from a culture of bacteria,yeast, mammalian cells, insect cells, plant cells, or the like,transformed to express a cDNA encoding the luciferase, or from an invitro cell-free system for making the luciferase from a nucleic acidencoding same. Luciferases are available from Promega Corporation,Madison, Wis.

The term “pyrophosphatase,” unless specified otherwise, refers to anyagent such as an enzyme (naturally occurring or mutant) that is capableof breaking down or hydrolyzing pyrophosphate that is generated duringthe course of a reaction, already present in the reaction mixture, orintroduced into a reaction mixture. The agent should be added at aconcentration sufficient to either catalyze the hydrolysis ofpyrophosphate in the reaction mixture at a rate that will preventaccumulation of pyrophosphate or prevent accumulation of pyrophosphatein any other manner. The amount of agent needed is readily determined bystandard procedures. While inorganic pyrophosphatases (hydrolyases) arepreferred agents in practicing this invention, there are many enzymetypes (e.g., transferases, kinases, and synthetases) that may also beused in the practice of the invention. See, for example, U.S. Pat. No.6,291,164 which is incorporated by reference in its entirety. A numberof such enzymes have been cloned and expressed in a recombinant host.See, for example, Ladror, U. S. et al., J. Biol. Chem. 266:16550-16555(1991) (Pyrophosphate: fructose-6-phosphate 1-phosphotransferase); Leyh,T. S. et al., J. Biol. Chem. 263:2409-2416 (1988) (ATP: sulfateadenylyltransferase); Leyh, T. S. et al., J. Biol. Chem. 267:10405-10410(1992) (ATP: sulfate adenylyltransferase); Weissbom, A. C., et al., J.Bacteriology 176:2611-2618 (1994) (UTP:glucose-1-phosphateuridylyltransferase); Allen, T. et al., Mol. Biochem. Parasitol. 74:99(1995) (Adenine phosphoribosyltransferase); Vonstein, V. et al., J.Bacteriol. 177:4540 (1995) (Orotate phosphotibosyltransferase); Charng,Y. Y. et al., Plant Mol. Biol. 20:37 (1992) (Glucose-1-phosphateadenylyltransferase); Kim, D. J. and Smith, S. M., Plant Mol. Biol.26:423 (1994) (Phosphoenolpyruvate carboxykinase); Jiang, Y. et al.,Exp. Parasitol. 82:73 (1996) (Hypoxanthine-guaninephosphoribosyltransferase); Pla, J. et al., Gene 165:115 (1995) (ATPphosphoribosyltransferase); Feldman, R. C. et al., Infect. Immun. 60:166(1992) (Uracil phosphoribosyltransferase); Vinitsky, A., J. Bacteriol.173:536 (1991) (Micotinate phosphoribosyltransferase); Ludin, K. M. etal., Curr. Genet. 25:465 (1994) (Amidophosphoribosyltransferase); Rose,A. B. et al., Plant Physiol. 100:582 (1992) (Anthranilatephosphoribosyltransferase); Hughes, K. T. et al., J. Bacteriol. 175:479(1993) (Quinolate phosphoribosyltransferase); Jagadeeswaran, P. et al.,Gene 31:309 (1984) (Xanthine-guanine phosphoribosyltransferase);Nakagawa, S., Biosci. Biotech. Biochem. 59:694 (1995) (FMNadenylyltransferase); Marolda, C. L. and Valvano, M. A., J. Bacteriol.175:148 (1993) (Mannose-1-phosphate guanylyltransferase); Kalmar, G. B.,Proc. Natl. Acad. Sci. USA 87:6029 (1990) (Choline phosphatecytidylyltransferase); Muller-Rober, B. et al., Plant Mol. Biol. 27:191(1995) (Glucose-1-phosphate adenylyltransferase); Sharmugam, K. et al.,Plant Mol. Biol. 30:281 (1996) (tRNA nucleotidyltransferase); Zapata, G.A. et al., J. Biol. Chem. 264:14769 (1989) (Acylneuraminatecytidylyltransferase); and Vakylenko, S. B. et al., Antiobiot.Khimioter. 38:25 (1993) (Gentamycin 2′-nucleotidyltransferase). If suchenzymes are used, it may be necessary to also employ a substrate whichis capable of either accepting a phosphate radical to give aphosphorylated product from pyrophosphate or effecting transfer of apyrophosphate radical when in the presence of the enzyme.

In one embodiment of the invention, luciferin derivatives are providedthat are substrates of CYP450 and are pro-substrates of luciferase. Whenthese luciferin derivatives are exposed to certain CYP450 isoforms,these isoforms metabolize the derivatives into compounds that can bereadily detected in a light-emitting reaction in the presence of theenzyme luciferase. In the absence of CYP450, the luciferin derivativesmay bind to luciferase, however they are not turned over as substrate inlight-generating reactions. In practicing the invention, the luciferinderivatives of the invention preferably have the following formula:

wherein R₁ represents hydrogen, hydroxyl, amino, C₁₋₂₀ alkoxy,substituted C₁₋₂₀ substituted alkoxy, C₂₋₂₀ alkenyloxy, C₂₋₂₀substituted alkenyloxy, C₁₋₂₀ halogenated alkoxy, C₁₋₂₀ substitutedhalogenated alkoxy, C₃₋₂₀ alkynyloxy, C₃₋₂₀ substituted alkynyloxy,cycloalkoxy, substituted cycloalkoxy, cycloalkylamino, substitutedcycloalkylamino, C₁₋₂₀ alkylamino, C₁₋₂₀ substituted alkylamino, C₁₋₂₀substituted alkylamino C₁₋₂₀ dialkylamino, or C₁₋₂₀ substituteddialkylamino, C₂₋₂₀ alkenylamino, C₂₋₂₀ substituted alkenylamino, C₂₋₂₀dialkenylamino, C₂₋₂₀ substituted dialkenylamino, C₂₋₂₀alkenylalkylamino, or C₂₋₂₀ substituted alkenylalkylamino, C₃₋₂₀alkynylamino, C₃₋₂₀ substituted alkynylamino, C₃₋₂₀ dialkynylamino,C₃₋₂₀ substituted dialkynylamino, C₃₋₂₀ alkynylalkylamino, C₃₋₂₀substituted alkynylalkylamino, C₃₋₂₀ alkynylalkenyl amino, or C₃₋₂₀substituted alkynylalkenylamino;

R₂ and R₃ independently represents C or N;

R₄ and R₅ independently represents S; O; NR₅ wherein R₈ representshydrogen or C₁₋₂₀alkyl; CR₉R₁₀ wherein R₉ and R₁₀ independentlyrepresent H, C₁₋₂₀ alkyl, or fluorine;

R₆ represents COR₁₁ wherein R₁₁ represents H, OH, C₁₋₂₀ alkoxide, C₂₋₂₀alkenyl, CH₂OH, or NR₁₂R₁₃ wherein R₁₂ and R₁₃ are independently H orC₁₋₂₀ alkyl; and

R₇ represents H, C₁₋₆ alkyl, C₁₋₂₀ alkenyl, halogen, or C₁₋₆ alkoxide.

In practicing this invention, particularly preferred luciferinderivatives include luciferin 6′ methyl ether (Luc ME), luciferin 6′ethyl ether (Luc EE), luciferin 6′ chloroethyl ether (Luc CEE),luciferin 6′ benzyl ether (Luc BE), luciferin 6′ 3-picolinyl ether (Luc3PE) and 6′ deoxyluciferin (H Luc).

In another embodiment of the invention, a method is provided formeasuring the activity of a cytochrome P450 enzyme. A luminogenicmolecule that is a P450 substrate and a bioluminescent enzymepro-substrate is contacted with one or more cytochrome P450 enzymes andbioluminescent enzyme, either simultaneously or in a stepwise manner,for a predetermined time. In the presence of P450, the luminogenicmolecule is metabolized into a substrate for the bioluminescent enzymein a first reaction. The bioluminescent enzyme then acts on thesubstrate in a second light emitting reaction. Cytochrome P450 activityis determined by measuring the amount of luminescence that is generatedfrom reaction mixture relative to a control (e.g., no P450 enzyme). Forthe P450 reaction to occur, P450 reductase, NADPH and Mg⁺² are generallypresent in the system. Similarly, the presence of ATP and Mg⁺² isgenerally necessary for firefly luciferase activity but not for Renillaluciferase activity. Any suitable concentration of luminogenic moleculemay be employed in the reaction mixture. In practicing this invention,the concentration of the luminogenic molecule generally ranges betweenabout 10 nM to 1 mM, preferably in the linear range of the substratedose response by a particular P450 isoform, most preferably at the Kmfor the particular substrate/P450 isoform reaction or at Vmax for thatreaction.

The invention also provides a method for determining P450 activity basedon luminogenic molecules that are natural coelenterazine andcoelenterazine derivatives (collectively referred to ascoelenterazines). The P450 acts on these luminogenic molecules in one oftwo ways. In one reaction pathway, the luminogenic molecules are P450substrates and bioluminescent enzyme pro-substrates and do not exhibitthe characteristic coelenterazine chemiluminescent (luminescence in theabsence of a bioluminescent enzyme, e.g. Renilla-type luciferase). P450metabolism of the luminogenic molecule in a first reaction generates thesubstrate for the Renilla luciferase. The Renilla luciferase then actson the substrate in a second light-emitting reaction. P450 activity isthen ascertained by measuring the luminescence of the reaction mixturerelative to a control reaction mixture. In the second reaction pathway,coelenterazine or coelenterazine derivatives exhibit chemiluminescenceand are substrates for Renilla-type luciferase. P450 metabolism of sucha luminogenic molecule results in the loss of chemiluminescence andactivity with Renilla-type luciferase. In both types of reactionpathways, P450 activity may be detected either directly by a change inchemiluminescence by the action of the P450 alone or indirectly by achange in bioluminescence from a Renilla-type luciferase. Useful, butnon-limiting, coelenterazines are disclosed in U.S. patent applicationSer. No. 10/053,482, filed Nov. 2, 2001, the disclosure which is herebyincorporated by reference in its entirety.

Luciferases differ somewhat in the ranges of conditions, of pH, ionicstrength, temperature, ATP concentration, magnesium ion concentration,luciferin concentration, and the like, over which they are active in theluciferase-luciferin reaction. Likewise, cytochrome P450 enzymes differsomewhat in the ranges of conditions, of pH, ionic strength,temperature, cofactor requirements, substrate concentration, and thelike, over which they are active in. It is, however, a simple matter fora skilled artisan to ascertain such ranges, and even the optimum ranges,for any particular luciferase and any particular cytochrome P450 enzyme.In practicing this invention, the amount of luciferase enzyme that maybe employed in a reaction mixture generally ranges between about 0.1microgram/mL to about 200 microgram/mL, preferably about 0.5microgram/mL to about 100 microgram/mL. The amount of P450 enzyme thatmay be employed in a reaction mixture generally ranges between about 0.1picomoles to about 200 picomoles, preferably about 0.4 to about 80.0picomoles.

The skilled artisan is also aware that compositions other than thosespecifically recited above will be or may be present in any assayreaction mixture, in order to, for example, maintain or enhance theactivity of the enzyme or as a consequence of the procedures used toobtain the aliquot of sample being subjected to the assay procedures.Thus, typically buffering agents, such as Tricine, HEPPS, HEPES, MOPS,Tris, glycylglycine, a phosphate salt, or the like, will be present tomaintain pH and ionic strength; a proteinaceous material, such as amammalian serum albumin (preferably bovine serum albumin) or lactalbuminor an ovalbumin, that enhances the activity of luciferases in theluciferase-luciferin reaction, may be present; EDTA or CDTA(cyclohexylenediaminetetraacetate) or the like, may be present, tosuppress the activity of metal-containing proteases or phosphatases thatmight be present in systems (e.g., cells) from which luciferase to beassayed is extracted and that could adversely affect the luciferase orthe ATP. Glycerol or ethylene glycol, which stabilize luciferases, mightbe present. Similarly, detergents or surfactants, particularly non-ionicdetergents, such as those of octoxynol (e.g., sold under the trademark“Triton X” of Rohm & Haas, such as Triton X-100) might be included,typically as remnants, carried into a solution used in an assayaccording to the invention, of a solution used to lyse cells from whichluciferase is extracted for the assay. Counterions to the magnesiumwill, of course, be present; as the skilled will understand, thechemical identities and concentrations of these counterions can varywidely, depending on the magnesium salt used to provide the magnesiumion, the buffer employed, the pH of the solution, the substance (acid orbase) used to adjust the pH, and the anions present in the solution fromsources other than the magnesium salt, buffer, and acid or base used toadjust pH. In practicing this invention, MgSO₄ or MgCl₂ are thepreferred sources of magnesium ion. In one procedure, the magnesium ioncan be supplied as the carbonate salt, to provide the desired magnesiumion concentration, in a solution with the buffer to be used (e.g.,Tricine) and then the pH of the buffered solution can be adjusted byaddition of a strong acid, such as sulfuric, which will result in lossof most of the carbonate (and bicarbonate) as carbon dioxide andreplacement of these anions with sulfate, bisulfate, Tricine anion, andpossibly also other types of anions (depending on other substances(e.g., phosphate salts) that provide anions and might be present in thesolution). Oxygen-diffusion from the air into the solution in which theassay method is carried out is sufficient to provide the molecularoxygen required in the P450 and luciferase-luciferin reactions. In anycase, it is well within the skill of the ordinarily skilled to readilyascertain the concentrations of the various components in an assayreaction mixture, including the components specifically recited above inthe description of the method, that are effective for activity of theP450 enzyme in the P450 reaction and luciferase in theluciferase-luciferin reaction.

The luminescence of the luciferin-luciferase reaction may be measuredusing a commercially available luminometer, a scintillation counter, aphotomultiplier photometer or a photoemulsion film. In one aspect ofthis invention, a one-step method for measuring P450 activity isprovided.

In the one-step method, the luminogenic molecule is contacted with boththe cytochrome P450 enzyme and the bioluminescent enzyme simultaneouslyor contemporaneously and the mixture is allowed to incubate for apredetermined time period. The cytochrome P450 metabolizes theluminogenic molecule into a substate for the bioluminescent enzyme in afirst reaction. The bioluminescent enzyme then acts on the substate in asecond light emitting reaction. Cytochrome P450 activity is indirectlydetermined by measuring the amount of luminescence that is generatedfrom the assay mixture relative to a control mixture. Controls mayinvolve replacement of P450 enzyme with water or the P450 buffer,replacement of recombinant P450 membrane preparation with a similarpreparation that lacks P450 enzyme, elimination of NADPH, or heatdenaturation of P450 enzyme prior to addition of the luciferinsubstrate. Luminescence can be measured after a predetermined incubationtime period or continuously from the time the reaction is initiated. Forinstance, the assay results shown in FIGS. 6 and 7 were readcontinuously from the time the reaction was initiated.

In another and preferred aspect of this invention, a two-step method formeasuring P450 activity is provided. In the two-step method, theluminogenic molecule is first incubated with the cytochrome P450 enzymefor a first predetermined time period. The P450 enzyme metabolizes theluminogenic molecule and converts it into a substrate for thebioluminescent enzyme. Thereafter, the reaction mixture containing theP450 enzyme and substrate is contacted with a bioluminescent, e.g.,luciferase, enzyme for a second predetermined time period. Thebioluminescent enzyme acts on the substrate in a second light emittingreaction. Cytochrome P450 activity is then indirectly determined bymeasuring the amount of luminescence that is generated from the reactionmixture relative to a control (e.g., no active P450 enzyme).

In practicing this aspect of the invention, a detergent, preferablynon-ionic, is preferably added to the P450/luminogenic molecule mixturejust prior to or at the same time as contact with the bioluminescent,e.g., luciferase, enzyme in the second step of the two-step reactionsystem. The detergent quenches the P450 enzyme without interfering withthe luciferase enzyme, thus allowing the analyst to measure luciferin(or luciferin derivative metabolite(s)) concentration dependentluminescence at the time of stopping the reaction without the complexityof having luciferin continuously added to the pool by an active P450enzyme. Moreover, the non-ionic detergent stimulates the luciferase andresults in a somewhat brighter reaction. In the case where a testcompound such as a drug is a luciferase inhibitor, the non-ionicdetergent diminishes the inhibitory effect on the luciferase and thusoffers an advantage to the analyst who is interested only in the effectsof the test compound on P450 activity. Suitable, but non-limiting,detergents include Tergitol (non-ionic); Brij 35 (non-ionic); Brij 58(non-ionic); Polymixin B; Triton X-100 (non-ionic); Triton X-305(non-ionic); Triton N101 (non-ionic); Chaps (zwitterionic); Chapso(zwitterionic); Bigchap (non-ionic); Thesit (non-ionic); Pluronic L64(non-ionic); Rhodasurf 870; Chemal LA-9; Sulfonyl 465; Deoxycholate(anionic); and CTAB (cationic). In practicing this invention, Tergitoltype NP-9, a polyglycol ether non-ionic surfactant, is preferred. Theamount of detergent present in the assay mixture generally rangesbetween about 0.03% to about 2.0%, preferably between about 0.1% toabout 1.0%.

For the one-step system and the first step of the two-step reactionsystems, the reaction mixture generally included the followingcomponents: 25 mM KPO₄ at pH 7.4 for CYP2C9, 100 mM KPO₄ at pH 7.4 forCYP1A2 etc. Other components were 1.3 mM NADP+, 3.3 mMglucose-6-phosphate, 0.4 units/mL glucose-6-phosphate dehydrogenase, 3.7mM MgCl₂. For the one-step system, 200 micromolar ATP is also used. Forthe second step (luciferase reaction) of the two-step system, 5 mM to200 mM Tricine buffer, preferably 20 mM to 200 mM Tricine buffer, isused although HEPES, HEPPS, MOPS, Phosphate, and Bicine buffers are alsouseful. Other components include 0.7 mM to 7.1 mM MgSO₄ (or MgCl₂), and0.1 micromolar to 1 mM, preferably 10 micromolar to 250 micromolar, ATP.For the one-step system and the first step of the two-step reactionsystem, the reactions are generally carried out at about 20° to 42° C.,preferably about 22° to 37° C. For the second step of the two-stepreaction system, the reactions are generally carried out at atemperature of about 4° to 60° C., preferably about 200 to 42° C. Whileany suitable predetermined time may be used for the one-step or two-stepreaction systems, the predetermined time for the one-step reactionsystem and the first step of the two-step reaction system generallyranges between about 1 minute to about 18 hours, usually about 1 minuteto about 4 hours (for the one step reaction system) or about 10 minutesto about 1 hour (for the first step of the two-step reaction system).For the second step of the two-step reaction system, luminescence isdetermined immediately to about 18 hours, preferably immediately toabout 3 hours after initiation of the luciferase reaction.

In another aspect of this invention, a reagent, method and kit forstabilizing luminescence-based reactions is provided. It has beendiscovered that the use of certain luciferase stabilizing molecules suchas reversible inhibitors of luciferase may provide a protective effectagainst the known self-catalyzed auto-degradation of the luciferaseenzyme, thus prolonging the luminescence signal and facilitating batchprocessing of reaction mixtures. As defined herein, a luciferasestabilizing agent is any molecule or group of molecules thatsubstantially stabilizes luciferase against self-catalyzedauto-degradation of luciferase by slowing down the bioluminescencereaction. Without the stabilizing agent, the luminescent signalhalf-life may be as low as a few minutes, e.g., about three minutes.When stabilizing agent is used, the signal half-life of the luciferasedbased reaction can be extended by as long as two hours or longerincluding overnight, depending on the choice of stabilizing agent andthe concentration used. Examples of luciferase stabilizing moleculesinclude, without limitation, reversible inhibitors of luciferase.Bioluminescent signals may decay rapidly, especially in the case where afirefly luciferase is used. These enzymes are known to inactivate at arate that is positively correlated with the luminogenic reaction rate.While bright signals resulting from rapid luciferase reaction rates maybe desirable, the rapid inactivation at high rates imposes a practicallimitation on the assay. A stable luminescent signal would facilitatereading multiple samples in sequence without a significanttime-dependent decay between samples. To achieve a stable luminescentsignal, it is possible to slow the reaction rate and thereby diminishrate of signal decay. One method for slowing the luciferase reactionrate is to include a luciferase inhibitor that is competitive withluciferin. In this invention, 2-amino-6-methylbenzothiazole (AMBT) or2-(4-aminophenyl)-6-methylbenzothiazole (APMBT), both competiveinhibitors of firefly luciferase, were found to be particularly usefulin achieving a stable luminescent signal. Generally, the luciferasestabilizing agent is introduced after the completion of the P450reaction and may be introduced prior to or at the start of the secondstep of two-step luminescent CYP450 assays.

In practicing this aspect of the invention, any suitable amount ofluciferase inhibitor may be used that is effective to enhance thestability and prolong the lifetime of the luminescent signal relative tothe luminescent signal generated in a comparable luciferase-basedreaction mixture in the absence of the inhibitor. Preferably the amountof inhibitor should be such that the signal half-life is at least abouttwo hours or more to allow for sample batch processing. Generally, theamount of the inhibitor ranges from about 1 micromolar to about 1millimolar in the reaction mixture, preferably from about 1 micromolarand about 500 micromolar in the reaction mixture, more preferably fromabout 10 micromolar to about 200 micromolar in the reaction mixture, andmost preferably about 100 micromolar in the reaction mixture.

In another embodiment of the invention, test compounds such as candidatedrugs can be screened and evaluated for their activities as substratesof or regulators, either inhibitors or activators of a cytochrome P450enzyme by using the luminogenic molecules, e.g. luciferin derivatives ofthe present invention. A candidate drug may be determined to beregulator or a substrate of a cytochrome P450 enzyme by contacting acytochrome P450 enzyme with the candidate drug, under conditionssuitable for interaction therebetween, providing at least one luciferinderivative, under conditions that would, in the absence of an inhibitoror substrate of the cytochrome P450 enzyme, be suitable for interactionwith the cytochrome P450 enzyme, and detecting the presence ofluminescent signal of luciferin and/or a luciferin derivative metabolitein the presence of the luciferase, wherein luciferin and/or a luciferinderivative metabolite would be, in the absence of an inhibitor of thecytochrome P450 enzyme, the product of the reaction between thecytochrome P450 enzyme and the luciferin derivative. Such efficient P450substrates and regulators, as deemed appropriate by those of skill inthe art, may be removed from a screening library where such efficientcytochrome P450 substrates and regulators are not desired in theremainder of the screening for a candidate drug.

In one aspect of the invention, a method is provided to distinguishbetween a substrate and an inhibitor of cytochrome P450 enzymes.Typically, the candidate compound is incubated with at least onecytochrome P450 enzyme under conditions, which allow for metabolism ofthe candidate compound prior to providing the luminogenic molecule,e.g., luciferin derivative, under conditions that would, in the absenceof an inhibitor or substrate of the cytochrome P450 enzyme, be suitablefor interaction between the luciferin derivative and the cytochrome P450enzyme. Any luciferin produced by the P450 metabolism of the luciferinderivative would then interact with luciferase in a light-emittingsecond reaction. The resulting light emitting reaction is compared tothe one obtained from contacting a cytochrome P450 enzyme with thecandidate drug, under conditions suitable for interaction therebetween,providing at least one luciferin derivative, under conditions thatwould, in the absence of an inhibitor of the cytochrome P450 enzyme, besuitable for interaction between the luciferin derivative and thecytochrome P450 enzyme. Metabolism of the candidate drug by a cytochromeP450 enzyme reduces its concentration in the assay medium and may leadto an apparent loss of cytochrome P450 inhibitory activity compared toconditions without metabolism of the compound which would indicate itwas a substrate for the enzyme. An inhibitory compound that was notmetabolized would show equal potency, irrespective of the time ofaddition of the optical cytochrome p450 enzyme substrate.

In another aspect of the invention, the drug candidate is preferablycontacted first with the P450 enzyme for a first predetermined timeperiod. Thereafter, the mixture is contacted with the luminogenicmolecule, e.g., luciferin derivative and bioluminescent enzyme, e.g.,luciferase, simultaneously or contemporaneously and the mixture isallowed to incubate for a second predetermined time period. CytochromeP450 activity is determined by measuring the amount of luminescence thatis generated from the reaction mixture relative to a control (e.g., noP450 enzyme).

In yet another (and preferred) aspect of the invention, the drugcandidate is preferably incubated first with the P450 enzyme for a firstpredetermined time period to form a first mixture. Thereafter, the firstmixture is contacted with the luminogenic molecule, e.g., luciferinderivative, to form a second mixture that is allowed to incubate for asecond predetermined time period. The second mixture is then contactedwith a bioluminescent enzyme, e.g., luciferase, to form a third mixturewhich is allowed to incubate for a third predetermined time period.Thereafter, the P450 activity resulting from the interaction of theenzyme with the drug candidate is determined by measuring luminescenceafter the third predetermined time period relative to a control (e.g.,no drug) reaction.

Any suitable concentration ranges for compound screenings may be used inpracticing this invention. For primary library screening, theconcentration of test compounds used generally ranges from about 1 toabout 100 micromolar, usually about 10 micromolar. Secondary and furtherscreens of primary hit test compounds generally employ wider ranges toestablish the dose dependency of the response and depends, in part, onthe potency of the test compound.

In practicing this aspect of the invention, non-ionic detergent ispreferably added to the second mixture prior to the addition ofluciferase in order to denature or deactivate the P450 enzyme. Suitabledetergents and amounts are described above.

In another embodiment of the invention, a high throughput assay methodis provided for screening a plurality of compounds to determine theireffect on cytochrome P450 activity. The test compounds are contactedwith one or more types of P450 enzymes, the luminogenic molecule, e.g.,luciferin derivative, and bioluminescent enzyme, e.g., luciferase, for apredetermined period of time. Thereafter, the P450 activity resultingfrom the interaction of the P450 enzyme with the compounds aredetermined by measuring luminescence.

In one aspect of the invention, the compounds are preferably contactedfirst with the P450 enzymes for a first predetermined time period.Thereafter, the mixture is contacted with the luminogenic molecule,e.g., luciferin derivative and bioluminescent enzyme, e.g., luciferasesimultaneously or contemporaneously and the mixture is allowed toincubate for a second predetermined time period. Cytochrome P450activity is determined by measuring the amount of luminescence that isgenerated after the second predetermined time period relative to acontrol (e.g., non-P450 enzyme) reaction. In another (and preferred)aspect of the invention, the compounds are preferably contacted firstwith the P450 enzymes for a first predetermined time period to formfirst mixtures. Thereafter, the first mixtures are contacted with theluciferin derivative to form second mixtures that are allowed toincubate a second predetermined time period. The second mixtures arethen contacted with luciferase to form a third mixture which is allowedto incubate for a third predetermined time period. Thereafter, the P450activities resulting from the interaction of the enzyme with the testcompounds are determined by measuring luminescence of the reactionmixture relative to a control (e.g., no P450 enzyme) reaction mixture.In practicing this aspect of the invention, non-ionic detergent ispreferably added to the second mixture prior to the addition ofluciferase. Suitable detergents and amounts are described above.

For compound screening, P450 is contacted first with the test compoundfor a predetermined time period prior to addition of the luminogenicmolecule, e.g., luciferin derivative. Another approach would involvecontacting the P450 with the drug and luciferin simultaneously. Yetanother approach would involve contacting the P450 with the luciferinfirst for a predetermined time period prior to addition of the testcompound.

In another embodiment of the invention, a cell-based method is providedfor screening test compound to determine its effect on cytochrome P450activity of the cell. The test compound is contacted with a cell, theluminogenic molecule, e.g., luciferin derivative, and bioluminescentenzyme, e.g., luciferase, for a predetermined period of time.Thereafter, the P450 activity resulting from the interaction of the cellwith the compound is determined by measuring luminescence of thereaction mixture relative to a control (minus test compound) reactionmixture.

In one aspect of the invention, the compound is preferably contactedfirst with the cell for a predetermined time period. Thereafter, thecell is contacted with the luciferin derivative and luciferasesimultaneously or contemporaneously and the cell is allowed to incubatewith the derivative and luciferase for a second predetermined timeperiod. Cytochrome P450 activity of the cell is determined by measuringthe amount of luminescence generated from the reaction mixture relativeto a control reaction mixture (e.g., minus test compound). In another(and preferred) aspect of the invention, the test compound is preferablycontacted first with the cell for a predetermined time period.Thereafter, the exposed cell is then contacted with the luciferinderivative and incubated for a second predetermined time period. Thecell is then contacted with luciferase to form a third mixture which isallowed to incubate for a third predetermined time period. Thereafter,the P450 activity of the cell resulting from the interaction of the cellwith the test compounds are determined by measuring luminescence of thereaction mixture relative to a control reaction mixture (e.g., minustest compound). In practicing this aspect of the invention, non-ionicdetergent is preferably added to the second mixture prior to or at thesame time as the addition of luciferase to ensure more complete releaseof luciferin or luciferin derivative metabolites(s), resulting in astronger signal and a more sensitive assay. Detergent will rupture thecells and release luciferin. In the absence of detergent, however,luciferin or luciferin derivative metabolite(s) may leak out of the celldue to its cell permeability and this would form the basis for areal-time live cell assay with luciferase and ATP in the medium.Suitable detergents and concentration ranges are described above.

In practicing this aspect of the invention, suitable cells would includeany cell that expresses one or more P450 enzymes and its requisitecofactors such as P450 reductase that utilitizes the luciferinderivatives. Such cells can be used to examine the effects of testcompounds on CYP450 enzyme activities present in the cell at the timethe test compound is applied. The cells can also be used to examine theeffects of test compounds on the expression of endogenous CYP450 genesor transgenes that encode CYP450s with gene regulatory sequences. Inthis case test compound-induced changes in gene expression can bedetected by measuring changes in the level of p450 enzyme activity.Representative examples include: (a) Primary hepatocytes from human oranimal sources (commercially available from several sources: Gentest,Woburn, Mass.; Clonetics, Inc., San Diego, Calif.; Xenotech LLC, Lenexa,Kans.); (b) Hepatocytic cell lines: HepG2, HepG2C3A. Commerciallyavailable from Amphioxus Cell Technologies Inc. (Houston, Tex.) and fromAmerican Type Culture Collection (ATCC), THLE-3 Commercially availablefrom ATCC, HepLiu porcine hepatocyte line, commercially available fromMultiCell Technologies (Warwick, R.I.), and BC2 human hepatoma cell line(Gomez-Lechon, M. J. et al (2001) “Expression and induction of a largeset of drug-metabolizing enzymes by the highly differentiated humanhepatoma cell line BC2”, Eur. J. Biochem. 268, 1448-1459); (c) Cellsexpressing recombinant P450s such as: HepG2 (Yoshitomi, S. et al (2001)“Establishment of the transformants expressing human cytochrome P450subtypes in HepG2, and their applications on drug metabolism andtoxicology”, Toxicol. In Vitro 15(3), 245-256), Chinese hamster ovary(CHO) cells (Gabelova, A. et al (2002) “Mutagenicity of7H-dibenzo(c,g)carbazole and its tissue specific derivatives ingenetically engineered Chinese hamster V79 cell lines stably expressingcytochrome p450”, Mutation Research, 517(1-2), 135-145), BEAS-2B, SV40immortalized human bronchial epithelial cells (Coulombe, R. A. et al(2002) “Metabolism and cytotoxicity of aflatoxin B1 in cytochromeP450-expressing human lung cells” J. Toxicol. Env. Health Part A,65(12), 853-867), MCL-5B lymphoblastoid cell line (commerciallyavailable from Gentest, Woburn Mass.); and (d) non-mammalian cells suchas yeast, bacterial, plant, fungal etc. with native expression ofP450(s) or the same having been transformed with an expressible P450cDNA(s) and P450 reductase.

For cell-based compound screening, any suitable amount of test compoundmay be used, depending on the potency of the test compound and toxicity.For primary library screening, the concentration of test compounds usedtypically ranges from about 1 to about 1000 micromolar, usually about10-100 micromolar. Any suitable incubation time may be used. Generally,the time period for incubation of a test compound with cells typicallyranges from about 1 minute to about 96 hours, usually about 24 hours toabout 72 hours.

Any suitable number of cells may be used for cell-based compoundscreening, including cell-based high throughput screening. A single cellwould be the low end of a general range while for a cell culturereceptacle surface with adherent cells, cell confluence or superconfluence would be considered to be the upper end of the general rangefor adherent cells. For suspension cultures, a cell-saturated suspensionwould be considered the upper end of a general range. In practicing thisinvention, the preferred number of cells would be the minimum number ofcells where signal is detectable above background to a maximum number ofcells described for the general range. For the incubation period, thesuitable temperature and pH ranges will depend on the requirements ofthe cell. Generally, most cells are cultured at pH 7.4 and at 37° C.,but most cells are viable, at least temporarily, over a range oftemperature of 4 to 42° C. and at pH ranges of about 6 to 9.

The cell-based luminescence detection assay can be performed in a numberof different ways. For instance, in one embodiment, luciferase and ATPmay be added to the cell medium and luminescence could be detecteddirectly as luciferin leaks out of the cells. In this case thetemperature, pH, salt concentration, and other growth requirements willdepend on the requirements of the cells. Luciferase is generally activeat the physiological salt, pH and temperature conditions typicallyemployed in cell culture.

In another embodiment of the cell-based luminescence detection assay,culture medium may be removed from the culture and added to a luciferasereaction. The conditions for this would be essentially as alreadydescribed for the second step of a two-step reaction assay describedabove.

In yet another embodiment of the cell-based assay of the invention, thecells may be lysed in an appropriate lysis buffer that contains ATP,luciferase along with detergent(s) to ensure lysis and then luminescenceis read immediately. For animal cells, a buffer with 0.1-1.0% non-ionicdetergents such as Triton X 100 or Tergitol is typically sufficient.Bacteria, plant, fungal or yeast cells are usually more difficult tolyse. Detergents, freeze/thaw cycles, hypotonic buffers, sonication,cavitation or combinations of these methods may be used. The method oflysis must produce a lysate that is compatible with luciferase activity.In the case where detergent is used, the detergent is one that iscompatible with active luciferase. Luminescence would be proportional tothe luciferin or luciferin derivative metabolite(s) generated by theP450 reaction and then released from the cells. In a representativeexample of cell-based luminescence detection, a volume of lysis bufferis added to an equivolume of the spent cell culture medium. In avariation of this embodiment of the invention, a cell lysate may beprepared with a lysis buffer that does not contain luciferase and ATP,then an aliquot of the lysate could be added to a one-step or two-stepluciferase-based assay as described above.

In yet another embodiment of the cell-based luminescence detection assayof the invention, a cell that either transiently or stably expresses arecombinant bioluminescent enzyme, e.g., luciferase, may be used. Anyconventional method for creating transient or stable transfected cellsmay be used. An expressible cDNA vector that has been introduced to thecell encodes the luciferase. Luminescence would then evolve as a P450(s)also present in the cell metabolizes the luciferin derivative suppliedin the medium. P450 activity may be determined by measuring luminescenceafter a predetermined time period either in situ directly or afterlysis. Vectors for making the cells by transient or stable transfectionare available from Promega (Madison, Wis.), Clontech (Pal Alto, Calif.)and Stratagene (La Jolla, Calif.).

In another embodiment of the invention, a tissue-based method isprovided for screening a test compound or library of compounds todetermine their effect on cytochrome P450 activity of the cell. The testcompound is contacted with tissue, the luminogenic molecule, e.g.,luciferin derivative, and bioluminescent enzyme, e.g., luciferase for apredetermined period of time. Thereafter, the P450 activity resultingfrom the interaction of the tissue with the compound is determined bymeasuring luminescence of the reaction mixture relative to a control(minus test compound) reaction mixture. Representative examples oftissue include, without limitation, liver, intestine, and lung. Thetissue may be used in the form of minces or slices such as liver slices.Generally, the same considerations provided above for cell-based assayswould be applicable.

In another embodiment of the invention, a method is provided forscreening a test compound to determine its effect on the cytochrome P450activity of an animal. The test compound and the luminogenic molecule,e.g., luciferin derivative, are administered to the animal. After apredetermined time period, a biological specimen is removed from theanimal. Representative biological specimen includes blood and serum,urine, feces, bile, cerebrospinal fluid, lymph, saliva, tears, andmucous (basically any body fluid where CYP450 metabolites might befound). Blood/serum, urine, feces and bile are preferable biologicalspecimens. Blood and feces would likely need to be processed. Forinstance, blood specimens may be processed to remove cells and produceserum. A fecal specimen may be processed to produce an extract. Most ofthe other fluids would ideally be added directly to a luciferase assayor may be optionally diluted prior to addition to the luciferase assay.The specimen is then contacted with luciferase, ATP and Mg²⁺. After apredetermined time period, P450 activity resulting from the interactionof the animal with the compound is determined by measuring luminescencerelative to a control. One type of control would include a specimenobtained from the animal prior to administration of the test compound.This type of control would involve administration of the luciferinderivative without exposure to the drug and the samples and measurementswould be taken at a set time. At a later time, after the luciferin iscleared from its system, the test experiment, luciferin derivative plusdrug exposure, would be performed on the same animal in the same way.This control has the advantage of using the same animal as test andcontrol. This principle could also be applied to cell-based and tissueslice assays. An alternative approach would be to have separate test andcontrol animals. The test animals would receive luciferin derivative anddrug while the control animals would receive only luciferin derivative

In one aspect of the invention, the compound and luciferin derivativeare preferably administered to the animal either simultaneously orcontemporaneously. After a first predetermined time period, a biologicalspecimen is obtained from the animal. The biological specimen is thencontacted with luciferase for a second predetermined time period. Theeffect of the test compound on cytochrome P450 activity of the animal isdetermined by measuring the luminescence produced from the reactionmixture relative to a control reaction mixture (e.g. specimen obtainedfrom control animals as described above).

In another aspect of the invention, the compound is preferablyadministered first to the animal. After a first predetermined timeperiod, the luciferin derivative is then administered to the animal.After a second predetermined time period, a biological specimen isobtained from the animal. The biological specimen is then contacted withluciferase for a predetermined time period. The effect of the testcompound on cytochrome P450 activity of the animal is determined bymeasuring the amount of luminescence that is generated from theluciferase reaction mixture relative to a control (e.g., based on aspecimen obtained from control animals as described above).

The test compounds and luminogenic molecules, e.g., luciferinderivatives, may be formulated by any suitable means and can be used astablets, capsules or elixirs for oral administration; suppositories forrectal administration; sterile solutions, suspensions for injectableadministration; and the like. Injectables can be prepared inconventional forms, either as liquid solutions or suspensions, solidforms suitable for solution or suspension in liquid prior to injection,or as emulsions. Suitable excipients are, for example, water, saline,dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate,cysteine hydrochloride, and the like. In addition, if desired, theinjectable pharmaceutical compositions may contain minor amounts ofnon-toxic auxiliary substances, such as wetting agents, pH bufferingagents, and the like. If desired, absorption enhancing preparations(e.g., liposomes) may be utilized.

The amount of the test composition required as an in vivo dose as wellas the amount of luciferin derivative used will depend on the route ofadministration, the type of animal being treated, and the physicalcharacteristics of the specific animal under consideration. The dose canbe tailored to achieve a desired effect, but will depend on such factorsas weight, diet, concurrent medication and other factors which thoseskilled in the medical arts will recognize.

In non-human animal studies, applications of potential drug candidatesare commenced at higher dosage levels, with dosage being decreased untilthe desired effect is no longer achieved or adverse side effectsdisappear. The dosage can range broadly depending upon the desiredaffects and the therapeutic indication. Typically, dosages may bebetween about 10 microgram/kg and 100 mg/kg body weight, preferablybetween about 100 microgram/kg and 10 mg/kg body weight. Administrationis preferably oral. Oral administration would be preferable, althoughabsorption through skin or mucous membranes, intravenous, subcutaneousor intraperitoneal injection routes may be used. While control of dosagemay be problematic in some instances, oral administration would allowthe analyst to examine first pass metabolism that is greatly influencedby p450s in the cells lining the gut where the compounds are absorbed.Administration by injection may be better for controlling dose inanimals.

In yet another aspect of the invention, an animal-based high throughputassay for screening compounds is provided. Laboratory animals are usefulfor defining mechanisms of drug activity and for testing therapeuticregimens. More recently, zebrafish and other teleosts have been found tobe particularly useful for in vivo high throughput screening ofcompounds that employ intact animals. As defined herein, the term“teleost” means of or belonging to the Telostei or Teleostomi, a groupconsisting of numerous fishes having bony skeletons and rayed fins.Teleosts include, for example, zebrafish, medaka, Giant rerio, andpuffer fish. See, for instance, U.S. Pat. No. 6,299,858 (Phylonix, Inc.,assignee), which is incorporated by reference in its entirety. Forinstance, test compound and luciferin derivative may be administered toliving embryonic teleosts contained in multi-well plates. The teleostsare maintained in a suitable medium such as water. After one or morepredetermined time periods, the medium is then contacted with luciferasefor a second predetermined time period. The effect of the test compoundon cytochrome P450 activity of the animal may be determined by measuringthe luminescence produced from the mixture relative to a controlreaction mixture absent test compound.

In another aspect of the invention, the compound is preferablyadministered first to the teleosts. After a first predetermined timeperiod, the animal is contacted with the luciferin derivative. After asecond predetermined time period, the medium is then contracted withluciferase. The effect of the test compound on cytochrome P450 activityof the animal may be determined by measuring the luminescence producedfrom the mixture relative to a control reaction mixture absent testcompound.

Transgeneic animals with a luciferase transgene are also useful inanimal-based assays for ascertaining P450 activity and for screening oneor more compounds. Luciferase transgenes have been efficiently expressedin livers, mostly in mouse. Xenogen, Inc. and others has developedtransgeneic animals such as mouse and rat with a luciferase transgenethat gets expressed in target tissues such as liver. See, for instance,U.S. Pat. Nos. 5,650,135 and 6,217,847, which are incorporated byreference in their entirety. Generally, such transgeneic animals areinjected with luciferin and imaging technology (in vivo biophotonicimaging) is then used to measure luminescence at the site of luciferaseexpression in the living, intact animal. Thus, in another aspect of theinvention, a transgenic animal having a bioluminescence enzyme, e.g.,luciferase, transgene may be administered a luminogenic substrate thatwill be converted to into a substrate for a bioluminescence enzyme intissues where the appropriate P450 is expressed. If the bioluminescenceenzyme, e.g., luciferase, transgene is also expressed in that tissue,light will be produced and such light may be detected by any suitablemeans. As discussed above, one or more drugs can be tested for P450effects in such transgeneic animals in a animal-based assay.Alternatively, tissue from such transgeneic animals can be used in atissue-based assay. A drug that inhibits P450 enzyme activity willdiminish the signal and a drug that induces P450 gene expression willenhance the signal.

The invention further provides a method and kit for relieving inhibitionof firefly luciferease by its inhibitor inorganic pyrophosphate (iPP).The presence of iPP in a luciferase-based reaction is undesirablebecause it may affect the reproducibility of the reaction. Pyrophosphateis a product of a luciferase-based reaction with ATP, O2 and luciferin.Under certain conditions, iPP may accumulate to inhibitory levels. Inaddition, components of a luciferase-based reaction may contributeinhibitory amounts of iPP. For example, orthophosphate salts used tobuffer the reaction may contain iPP as a contaminant. While avoiding theuse of phosphate buffered solutions can solve the iPP problem, this isoften inconvenient or impractical, particularly in P450 reactions wherephosphate buffers are generally used. It has been discovered that theinclusion of a pyrophosphatase such as an inorganic pyrophosphataseenzyme (iPPase) in the luciferase-based reaction reduces or preventsluciferase inhibition by iPP that may already be present in the assaymixture or that may be generated during the course of the luciferasereaction. While this discovery is generally applicable to allluciferase-based reactions where potential inhibition of luciferase byiPP may be a problem, the inclusion of a pyrophosphatase such as iPPasein luminescent cytochrome P450 (P450) assays has been found to beparticularly useful. Without being bound by any theory of operation, itis believed that the pyrophosphatase, e.g., iPPase, acts to prevent thebuild-up of iPP as well as remove it from a solution by hydrolyzing theiPP into orthophosphate.

In practicing this invention, the pyrophosphatase, e.g., iPPase, may beadded prior to, simultaneously with, or shortly after luciferaseaddition. Preferably, the pyrophosphatase, e.g., iPPase, is added to theassay mixture simultaneously with luciferase addition to degrade any iPPalready present in the mixture and generated during the luciferasereaction. The amount of pyrophosphatase, e.g., iPPase, used in thereaction is sufficient to remove or eliminate any iPP that may alreadybe present or may be subsequently generated during the course of theluciferase reaction. Generally, in the presence of a pyrophosphatase,e.g., iPPase, the level of iPP in the reaction mixture is eliminated orreduced to an amount which has little or no significant effect onluciferase activity during a luciferase reaction. That is, the level ofiPP is low enough to reduce luciferase inhibition to an insignificantlevel. The iPPase may be derived from a variety of sources includingwithout limitation, yeast, prokaryotes and mammals. In practicing thisinvention, iPPase derived from Saccharomyces cerevisiae is preferred.

In one embodiment of this invention, the pyrophosphatase, e.g., iPPase,may be included in a cell-free or celled-based luminescent P450 assay.P450 enzymes convert luciferin derivatives that are pro-substrates forfirefly luciferase into luciferin substrates that then react withluciferase to generate light. For instance, in the first step of thetwo-step method described above, a P450 enzyme may be incubated with aluciferin derivative under suitable assay reaction conditions. In asecond step, the luciferin substrate generated in the first step is thendetected luminogenically when luciferase and ATP are added to themixture of the first step. Generally, it is convenient to employ KPO₄buffers in the first step to buffer pH and to provide optimal saltconcentration for specific P450 isoforms by varying its concentration.However, some KPO₄ buffers carry sufficient amounts of iPP contaminantwhich may inhibit luciferase and thus impair luciferin detection. Byadding an iPPase to the reaction mixture, inhibition by the presence ofany iPP contaminant may be reduced or prevented.

In another embodiment of this invention, the pyrophosphatase, e.g.,iPPase, may be included in any luciferase reaction, including cell-freeor cell-based luciferase assays. The addition of pyrophosphatase, e.g.,iPPase, as a reaction component would allow these assays to be performedin conventional phosphate buffers without concern for iPP inhibition ofluciferase. Other applications may include the use of iPPase inluciferase assays where the iPP generated by the luciferase reactionaccumulates to inhibitory levels.

In another embodiment of the invention, a kit is provided fordetermining the activity of a CYP450 or the effect of a substance, e.g.,a drug candidate, on cytochrome P450 enzyme activity. Such kitscomprise, in one or more containers, usually conveniently packaged tofacilitate use in assays, quantities of various compositions essentialfor carrying out the assays and methods in accordance with theinvention. Thus, the kit may include one or more types of cytochromeP450 enzymes; one or more luminogenic molecules, e.g., D-luciferinderivatives, that are substrates for one or more types of cytochromeP450 enzymes and a pro-substrate of a bioluminescent enzyme, e.g.,luciferase enzyme; a bioluminescent enzyme, e.g., luciferase enzyme; anddirections for using the kit. Optionally, the kit includes ATP, a sourceof Mg ions, non-ionic detergent, a pyrophosphatase, e.g., iPPase, and/orbuffers or any other reaction components to provide a solution atsuitable pH and ionic strength. As indicated, the various components canbe combined, e.g., in solution or a lyophilized mixture, in a singlecontainer or in various combinations (including individually) in aplurality of containers The preferred kit includes vial(s) with P450substrate (e.g., D-luciferin derivative), vial(s) containing a mixtureof bioluminescent enzyme, e.g., luciferase, and optional iPPase(preferably from Saccharomyces cervisiae) (preferably a lyophilizedpreparation) and a vial with a dilution buffer that contains ATP for thebioluminescent enzyme, e.g., luciferase (in the case of a lyophilizedluciferase/ATP preparation, this buffer is a “reconstitution buffer”).The dilution buffer or reconstitution buffer would also contain thedetergent.

The kits can also include, as well known to the ordinary skilled in theart, various controls and standards, including no P450 (negativecontrol), to ensure the reliability and accuracy of the assays carriedout using the kits.

EXAMPLES Materials

Sf9 cell expressed CYP450 preparations (Supersomes™), pooled human livermicrosomes and NADPH generating system (NADP+, glucose-6-phosphate andglucose-6-phosphate dehydrogenase) were purchased from GenTest (Woburn,Mass.). The substrates, luciferin 6′ methyl (Luc ME), ethyl (Luc EE),chloroethyl (Luc CEE) and benzyl (Luc BE) ethers and dehydroxyluciferin(H-Luc) were manufactured by Promega Biosciences (San Luis Obispo,Calif.). Luc ME and Luc EE were also purchased from Sigma-Aldrich (St.Louis). The recombinant, mutant of firefly luciferase from Photurispennsylvanica was from Promega (17). All chemical reagents and solventsreferred to herein are readily available from a number of commercialsources including Aldrich Chemical Co. or Fischer Scientific. NMRspectra were run on a Hitachi 60 MHz R-1200 NMR spectrometer or a VarianVX-300 NMR spectrometer. JR spectra were obtained using a Midac M seriesFT-IR instrument. Mass spectral data were obtained using a Finnegan MAT90 mass spectrometer. All melting points are corrected.

Example 1 Synthesis of Luciferin Derivatives

(a) Preparation of 2-cyanobenzothiazole Derivatives

Luciferol:

A suspension of D-luciferin free acid (0.43 g, 1.53 mmol) in THF (15 mL)was cooled in a −20° C. bath (dry ice-isopropanol). To the suspensionwas added dropwise via syringe a solution of borane-THF (1.8 mL of a 1 Msolution in THF, 1.8 mmol). The pale yellow solution was allowed to warmto ambient temperature overnight (about 15 h) under nitrogen. Additionalborane-THF (2.5 mL of a 1 M solution in THF, 2.5 mmol) was added and thereaction went an additional 24 h. The excess borane-THF was quenched bythe addition of 10% aqueous acetic acid solution and the resultingbilayer was extracted with ethyl acetate (3×75 mL). Combined extractswere dried and evaporated to give an orange solid that was purified bychromatography on silica gel (70 g) using 4:1 dichloromethane-methanol.This operation separated the remaining D-luciferin free acid from a lesspolar product mixture. The less polar product mixture was separated byreverse-phase HPLC on a 1-inch Synergi 4μ MAX-RP 80A column (100×21.20mm) using a methanol-water gradient. Appropriate fractions were pooledand evaporated to provide 10 mg (3% yield) of the desired product as apale yellow solid. This product was 95.4% pure by HPLC analysis. MS(ESI−): m/z 264.4 (M−H)⁻; calc'd: 265.01.

2-Cyanobenzothiazole

A suspension of potassium cyanide (1.50 g, 23.0 mmol) indimethylsulfoxide (DMSO, 100 mL) was prepared in a 1-L 3-necked flaskfitted with a reflux condenser, a heating mantle and an internaltemperature probe. The 2-chlorobenzothiazole (2.6 g, 2.0 mL, 15.3 mmol)was added to the reaction flask via pipet and the reaction was heated at80° C. (internal temperature). The reaction was monitored periodicallyby TLC (9:1 heptane-ethyl acetate). After 5 hours the reaction appearedto be about 50% complete. After 17 hours the reaction was judgedcomplete by TLC analysis. The reaction mixture was allowed to cool toroom temperature and was then extracted with ether (5×100 mL). Theextracts were dried over sodium sulfate and concentrated to give ayellow-orange solid. The crude solid was purified on silica gel (100 g)using 9:1 heptane-ethyl acetate. Fractions containing product werepooled and concentrated to give 1.4 g (57%) of the desired product as ayellow-orange solid. The structure was confirmed by ¹H NMR analysis inDMSO-d6.

6-(2-Chloroethoxy)-2-cyanobenzothiazole

A suspension of 2-cyano-6-hydroxybenzothiazole (1.0 g, 5.68 mmol) inacetone (5 mL) was prepared in a 50-mL 2-necked round-bottomed flaskfitted with a reflux condenser. Anhydrous potassium carbonate (1.57 g,11.3 mmol) was added to the reaction mixture and the suspension turnedto a yellow solution. Then 1-bromo-2-chloroethane (1.06 g, 0.56 mL, 7.38mmol) was added by syringe and the reaction mixture was heated at refluxusing a heated stir plate and oil bath. The reaction was monitoredperiodically by RP HPLC. After 5 hours the reaction appeared to be about30% complete. Additional 1-bromo-2-chloroethane was added (2.84 mmol,0.21 mL) and the reaction was refluxed overnight. HPLC analysisindicated the reaction was about 50% complete. Additional1-bromo-2-chloroethane (2.84 mmol, 0.21 mL) and potassium carbonate(0.60 g, 4.37 mmol) were added and the reaction was refluxed for therest of the day (about 7 h). The reaction was about 64% complete, and itwas refluxed overnight. The next morning the reaction was found to beabout 84% complete. Additional 1-bromo-2-chloroethane (2.84 mmol, 0.21mL) and potassium carbonate (0.60 g, 4.37 mmol) were added and thereaction was refluxed for the rest of the day (about 7 h). At this pointthe reaction was about 92% complete. The reaction mixture was allowed tocool to room temperature and it was then filtered through glass fiberpaper and rinsed with acetone. The filtrate was concentrated to give ayellow-brown solid. The crude solid (1.3 g) was purified on silica gel(100 g) using 4:1 heptane-ethyl acetate. Fractions containing productwere pooled and concentrated to give 1.0 g (74%) of the desired productas an off-white solid. The structure was confirmed by ¹H NMR analysis inDMSO-d6.

6-(4-Trifluoromethylbenzyloxy)-2-cyanobenzothiazole

A suspension of 2-cyano-6-hydroxybenzothiazole (1.0 g, 5.68 mmol) inacetone (5 mL) was prepared in a 100-mL 2-necked round-bottomed flaskfitted with a reflux condenser. Anhydrous potassium carbonate (0.94 g,6.82 mmol) was added to the reaction mixture and the suspension turnedto a yellow solution. Then 4-(trifluoromethyl)benzyl bromide (1.49 g,6.25 mmol) was added and the reaction mixture was heated at reflux for15 h using a heated stir plate and oil bath. The reaction mixture wasallowed to cool to ambient temperature and then filtered to removeinorganic salts. The filtrate was concentrated by rotoevaporation toprovide 1.7 g (89% yield) of an off-white solid that was used withoutpurification. The structure was confirmed by ¹H NMR analysis in DMSO-d6.

6-(Benzyloxy)-2-cyanobenzothiazole

A suspension of 2-cyano-6-hydroxybenzothiazole (0.35 g, 2.00 mmol) inacetone (35 mL) was prepared in a 100-mL 2-necked round-bottomed flaskfitted with a reflux condenser. Anhydrous potassium carbonate (0.33 g,2.40 mmol) was added to the reaction mixture and the suspension turnedto a yellow solution. Then benzyl bromide (0.41 g, 0.29 mL, 2.40 mmol)was added and the reaction mixture was heated at reflux for 15 h using aheated stir plate and oil bath. The reaction mixture was allowed to coolto ambient temperature and then filtered to remove inorganic salts. Thefiltrate was concentrated by rotoevaporation to provide 0.51 g of a paleyellow solid. The crude product was purified by flash chromatography onsilica gel (24 g) using a mobile phase consisting of a mixture of 4:1heptane-ethyl acetate to provide 410 mg (77% yield) of the desiredproduct as a white foam. The structure was confirmed by ¹H NMR analysisin DMSO-d6.

6-(2-Phenylethoxy)-2-cyanobenzothiazole

A suspension of 2-cyano-6-hydroxybenzothiazole (1.00 g, 5.68 mmol) inacetone (5 mL) was prepared in a 100-mL 2-necked round-bottomed flaskfitted with a reflux condenser. Anhydrous potassium carbonate (1.18 g,8.52 mmol) was added to the reaction mixture and the suspension turnedto a yellow solution. Then (2-bromoethyl)benzene (1.37 g, 0.88 mL, 7.38mmol) was added and the reaction mixture was heated at reflux for usinga heated stir plate and oil bath. Progress of the reaction was monitoredby HPLC analysis of reaction aliquots. After 15 h of reflux the reactionwas about 40% complete. Additional (2-bromoethyl)benzene (0.5equivalents, 2.84 mmol) and potassium carbonate (0.5 equivalents, 2.84mmol) were added periodically during the course of two days until thestarting material was consumed. The reaction mixture was allowed to coolto ambient temperature and then filtered to remove inorganic salts. Thefiltrate was concentrated by rotoevaporation to provide 2.2 g of a crudeoil. The crude product was purified by flash chromatography on silicagel using an initial mobile phase consisting of a mixture of 9:1heptane-ethyl acetate. The mobile phase was adjusted to a mixture of 5:1heptane-ethyl acetate, and then adjusted to a mixture of 7:3heptane-ethyl acetate to provide 800 mg (50% yield) of the desiredproduct as an off-white foam. The structure was confirmed by ¹H NMRanalysis in DMSO-d6.

6-(Geranyloxy)-2-cyanobenzothiazole

To a dry 25-ml round-bottomed flask containing acetone (5 mL), anhydrouspotassium carbonate (1.2 g, 8.4 mmole), and geranyl bromide (1.5 mL, 7.3mmole) was added 6-hydroxy-2-cyanobenzothiazole (1 g, 5.6 mmole). Themixture was refluxed under argon with stirring. Reaction progress wasmonitored by TLC analysis, developing with 2:1 heptane-ethyl acetate.After 20 h, the potassium carbonate was filtered from the cooledreaction mixture. The solution was concentrated in vacuo to yield 2.1 gof solid. The solid was further purified by flash chromatography using amixture of 9:1 heptane-ethyl acetate. Appropriate fractions were pooledand evaporated to yield 0.84 g of solid. The structure was confirmed by¹H NMR analysis in DMSO-d6.

6-(Prenyloxy)-2-cyanobenzothiazole

To a dry 25-ml round-bottomed flask containing acetone (5 mL), anhydrouspotassium carbonate (1.2 g, 8.4 mmol), and prenyl bromide (839microliters, 7.3 mmole) was added 6-hydroxy-2-cyanobenzothiazole (1.0 g,5.6 mmol). The mixture was refluxed under argon with stirring. Reactionprogress was monitored by TLC analysis, developing with 2:1heptane-ethyl acetate. After 28 h, the potassium carbonate was filteredfrom the cooled reaction mixture. The solution was concentrated in vacuoto yield 1.7 g of solid. The solid was further purified by flashchromatography using 9:1 heptane-ethyl acetate and gradually stepping to4:1 heptane-ethyl acetate. Appropriate fractions were pooled andevaporated to yield 0.8 g of solid. The structure was confirmed by ¹HNMR analysis in DMSO-d6.

6-(2-Picolinyloxy)-2-cyanobenzothiazole

(Bromomethyl)pyridine hydrobromide (1.87 g, 7.38 mmol) and2-cyano-6-hydroxybenzothiazole (1.00 g, 5.68 mmol) were suspended inacetone (50 mL). After introduction of potassium carbonate (1.96 g, 14.2mmol), the suspension was refluxed for 72 h under nitrogen. Aftercooling down the mixture and filtering the solids, the filtrate wasconcentrated and the residue was purified by silica gel chromatographyusing 50%-75% ethyl acetate in heptane. A yellowish solid was obtainedin 72% yield. MS (ESI+): m/z 267.90 (M+H)⁺; calc'd: 268.05.

6-(3-Picolinyloxy)-2-cyanobenzothiazole

To a solution of 2-cyano-6-hydroxybenzothiazole (0.39 g, 2.2 mmol) inacetone (50 mL) was added 3-(bromomethyl)pyridine hydrobromide (0.7 g,2.76 mmol), cesium carbonate (2.15 g, 6.6 mmol), and a catalytic amountof sodium iodide. After adding 3A molecular sieves, the yellowsuspension was refluxed for 40 h under nitrogen. After cooling down themixture and filtering the solids, the filtrate was concentrated and theresidue was purified by silica gel chromatography using 50%-100% ethylacetate in heptane. A yellowish solid was obtained in 61% yield. MS(ESI+): m/z 267.64 (M+H)⁺; calc'd: 268.05.

6-(4-Picolinyloxy)-2-cyanobenzothiazole

To a solution of 2-cyano-6-hydroxybenzothiazole (1.18 g, 6.71 mmol) inacetone (50 mL) was added 3 Å molecular sieves and cesium carbonate(3.98 g, 12.2 mmol). The resulting suspension was stirred at roomtemperature for two hours. Then another equivalent of cesium carbonate(1.99 g, 6.1 mmol) was introduced, followed by addition of4-(bromomethyl)pyridine hydrobromide (1.0 g, 6.1 mmol) and a catalyticamount of cesium iodide. The resulting yellow suspension was refluxedfor 48 h under nitrogen. After cooling down the mixture and filteringthe solids, the filtrate was concentrated and the residue was purifiedby silica gel chromatography, using 30% ethyl acetate in heptane toremove the starting material and then 25% methanol in ethyl acetate. Ayellowish solid was obtained in 70% yield. MS (ESI+): m/z 267.74 (M+H)⁺;calc'd: 268.05.

(b) General Procedures for the Conversion of 2-cyanobenzothiazoleDerivatives to D-luciferin Derivatives.

A solution of 0.39 M aqueous cysteine hydrochloride monohydrate (1.3equivalents, based on the quantity of the 2-cyanobenzothiazolederivative) was added dropwise to an equal volume of a 0.39 M solutionof potassium carbonate, maintaining the pH at 6-7 by addition of 6 MHCl. In a separate reaction flask the 2-cyanobenzothiazole derivativewas dissolved in sufficient methanol to prepare a 0.1 M solution. Thissolution was purged with nitrogen to remove oxygen. Thecysteine/potassium carbonate solution described above was added dropwiseto the reaction flask containing the 2-cyanobenzothiazole derivative,maintaining the pH at 6-7 by addition of 6 M HCl. The reaction wasmonitored by TLC, and when complete the reaction mixture wasconcentrated by rotoevaporation using a cold water bath (<30° C.).

6′-Deoxyluciferin (H-Luc)

Prepared from 2-cyanobenzothiazole (100 mg, 0.62 mmol) according to thegeneral procedure. The crude solid product was purified by flashchromatography on silica gel (20 g) using 9:1 dichloromethane-methanolto afford 163 mg (99%) of desired product as a pale yellow solid. Thismaterial was 96% pure by HPLC analysis. MS (ESI−): m/z 263.40 (M−H)⁻;calc'd: 262.99.

Luciferin 6′-(2-chloroethyl)ether (Luc CEE)

Prepared from 6-(2-chloroethoxy)-2-cyanobenzothiazole (1.0 g, 4.19 mmol)according to the general procedure. The solid product thus obtained was99.5% pure by HPLC analysis and further purification was not deemednecessary. The yield of this product was 1.36 g (95% yield). MS (ESI+):m/z 342.94 (M+H)⁺; calc'd: 343.00.

Luciferin 6′-benzyl ether (Luc BE)

Prepared from 6-(benzyloxy)-2-cyanobenzothiazole (0.41 g, 1.5 mmol)according to the general procedure. The crude solid product was purifiedby flash chromatography on silica gel (90 g) using 100% dichloromethaneinitially, gradually stepping up to 8:2 dichloromethane-methanol toafford 190 mg (34% yield) of desired product. MS (ESI−): m/z 368.67(M−H)⁻; calc'd: 369.04.

Luciferin 6′-(4-trifluoromethyl)benzyl ether (Luc TFMBE)

Prepared from 6-(4-trifluoromethylbenzyloxy)-2-cyanobenzothiazole (1.7g, 5.08 mmol) according to the general procedure. The resulting solidwas purified by flash chromatography on silica gel using initially amixture of 99:1 dichloromethane-methanol, gradually stepping up to 9:1dichloromethane-methanol. Appropriate fractions were pooled andevaporated to yield 700 mg (31%) of the desired product as a solid.

Luciferin 6′-(2-phenylethyl)ether (Luc PEE)

Prepared from 6-(2-chloroethoxy)-2-cyanobenzothiazole (0.80 g, 2.85mmol) according to the general procedure. The resulting solid waspurified by flash chromatography on silica gel using initially a mixtureof 6:3:1 heptane-ethyl acetate-methanol, then a mixture of 5:3:2methanol-heptane-ethyl acetate. Appropriate fractions were pooled andevaporated to yield 145 mg (14%) of the desired product as a solid. MS(ESI+): m/z 384.52 (M+H)⁺; calc'd 385.07.

Luciferin 6′-geranyl ether (Luc GE)

Prepared from 6-geranyloxy-2-cyanobenzothiazole (0.8 g) according to thegeneral procedure. The resulting solid was purified by flashchromatography on silica gel using 9:1 dichloromethane-methanol.Appropriate fractions were pooled and evaporated to yield 101 mg ofsolid. The structure was confirmed by ¹H NMR analysis in DMSO-d6.

Luciferin 6′-prenyl ether (Luc PE)

Prepared from 6-prenyloxy-2-cyanobenzothiazole (0.8 g) according to thegeneral procedure. The resulting solid was first purified by flashchromatography using 9:1 dichloromethane-methanol gradually stepping to8:2 dichloromethane:methanol. The solid was then repurified by flashchromatography using 2:1 heptane-ethyl acetate. Appropriate fractionswere pooled and evaporated to yield 339 mg of yellowish solid. Thestructure was confirmed by ¹H NMR analysis in DMSO-d6. Fluorescence HPLCanalysis indicated background luciferin, so the solid was furtherpurified by preparative reverse-phase HPLC.

Luciferin 6′-(2-picolynyl)ether (Luc 2PE)

Prepared from 6-(2-picolinyloxy)-2-cyanobenzothiazole (250 mg, 0.94mmol) according to the general procedure. The solid product thusobtained was 92.0% pure by HPLC analysis and the yield of this productwas 80%. MS (ESI+): m/z 371.55 (M+H)⁺; calc'd 372.04.

Luciferin 6′-(3-picolynyl)ether (Luc 3PE)

Prepared from 6-(3-picolinyloxy)-2-cyanobenzothiazole (250 mg, 0.94mmol) according to the general procedure. The solid product thusobtained was 99.8% pure by HPLC analysis and further purification wasnot deemed necessary. The yield of this product was 60%. MS (ESI+): m/z371.64 (M+H)⁺; calc'd 372.04.

Luciferin 6′-(4-picolynyl)ether (Luc 4PE)

Prepared from 6-(4-picolinyloxy)-2-cyanobenzothiazole (267 mg, 1.0 mmol)according to the general procedure except that 5 mL DMF was used todissolve the starting material. The solid product thus obtained was96.0% pure by HPLC analysis and further purification was not deemednecessary. The yield of this product was 20%. MS (ESI+): m/z 371.61(M+H)⁺; calc'd 372.04.

Example 2 Two step CYP450/Luciferase Reaction and Luciferin DerivativesEvaluation

In this Example, a procedure for a two-step CYP450/luciferase assay isprovided. Luciferin derivatives were evaluated as P450 substrates andluciferase pro-substrates using this procedure. 20 microliter CYP450reactions were prepared at pH 7.4 in an amount of KPO₄ buffer that isoptimal for a given CYP450 isoform (100 mM for CYP1A1, CYP1A2, CYP2B6,CYP2D6 and CYP2E1; 50 mM for CYP2C8 and CYP2C19; 25 mM for 2C9 and forpooled human liver microsomes; 200 mM for CYP3A4). For CYP2A6 100 mMTris at pH 7.5 was substituted for KPO₄. Reaction mixes also contained1.3 mM NADP+, 3.3 mM glucose-6-phosphate, 0.4 U/ml glucose-6-phosphatedehydrogenase, 3.3 mM MgCl₂ and a luciferin derivative/CYP450 substrate.Reactions were initiated by addition of 0.4 pmoles recombinant humanCYP450 co-expressed with CYP450 reductase in Sf9 cell microsomalmembranes or 4 microgram pooled human liver microsomes and incubation at37° C. After an initial incubation period at 37° C. 20 microliter CYP450reactions were mixed with an 80 microliter luciferase reaction mix. Theluciferase reaction mix contained 250 micromolar ATP, 5-25 microgram/mLthermostable luciferase from Photuris pennsylvanica prepared asdescribed in WO/9914336, published Mar. 25, 1999 which is incorporatedby reference in its entirety), 20 mM Tricine pH 7.8, 0.1 mM EDTA, 8 mMMgCl₂, 0.6 mM coenzyme A and 33 mM DTT. Assays were also performed using50 microliter CYP450 and luciferase reaction volumes (e.g., FIG. 3,panel B, table 3). Light output was measured immediately in a TurnerReporter, Turner 20/20 or Berthold Orion luminometer. Because thecalibration of instruments from different manufacturers varies thequantitation of light output is instrument-specific and directcomparisons between instruments cannot be made.

O-dealkylation and hydroxylation are common CYP450 catalyzed xenobiotictransformations (9). A panel of recombinant human CYP450 microsomepreparations was tested for O-dealkylase activity against luciferin 6′methyl (Luc ME), ethyl (Luc EE), chloroethyl (Luc CEE), benzyl (Luc BE),p-CF3 benzyl (Luc TFMBE), phenylethyl (Lluc PEE), geranyl (Luc GE), 2, 3and 4 picolinyl (Luc 2PE, Luc 3PE and Luc 4PE) and prenyl (Luc PE)ethers and for hydroxylase activity against dehydroluciferin (H-Luc)(FIG. 2). These compounds are either inactive in a light generatingluciferase reaction or only modestly active as compared to authenticluciferin. The CYP450 activities tested are contained in microsomefractions from insect cells that over-express a single recombinant humanCYP450 isoform in combination with NADPH CYP450 reductase. It wasreasoned that if CYP450s dealkylated or hydroxylated the luciferinderivatives at the 6′ position, authentic luciferin would be generatedthat could be detected enzymatically in a light generating fireflyluciferase reaction as described by the equation in FIG. 1.

Luc ME, Luc EE, Luc CEE, Luc BE, H-Luc, Luc TFMBE, Luc PEE, Luc GE, Luc2PE, Luc 3PE and Luc 4PE and Luc PE were subjected to a two-step assaywhere they were first incubated with a panel of CYP450 enrichedmicrosomes or control microsomes (with no detectable CYP450 activity)under conditions where the CYP450s are known to be active. The panelincluded CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19,CYP2D6, CYP2E1 and CYP3A4. After an initial incubation with CYP450,luciferase and its requisite cofactors were added and light output wasmonitored (FIGS. 3 and 9). Light output was increased significantly overcontrols by CYP1A2, CYP2C8 and CYP2C9 with Luc ME, CYP2C8, CYP2C9 andCYP3A4 with Luc BE, CYP1A1, CYP1A2, CYP2C8 and CYP2C9 with Luc EE, byCYP1A1, CYP1A2, CYP2C8 and CYP2C9 with Luc CEE, by CYP1A1, CYP2C8 andCYP2C9 by Luc TFMBE, by CYP3A4 with Luc PE, Luc PEE, by CYP1A1 with LucGE and by CYP3A4 with Luc PE (FIG. 3). With Luc 2PE, Luc 3PE and Luc 4PEthe most obvious increase in light output was with CYP1A1 and CYP3A4,the effect being most pronounced for both isoforms with Luc 3PE (FIG. 9)These isoforms apparently dealkylated luciferin 6′ alkyl ethers and 6′substituted alkyl ethers to form luciferin while the other isoformstested did not. When H-Luc was used as substrate CYP1A2 and CYP2C9increased light output over controls. H-Luc was apparently hydroxylatedto form luciferin and within the panel tested the reaction was mostsignificant with the CYP2C9 isoform. Values for isoform/substratecombinations not shown were similar to Sf9 cell membrane controls. Foreach of the panel screens, background luminescence reflects at least inpart the presence of contaminating D-luciferin in the unreactedpreparations of d-luciferin derivatives. Luminescent CYP1A1, CYP1A2,CYP2C8, CYP2C9 and CYP3A4 reactions were dose-dependent with respect tosubstrate (data not shown).

Example 3 Time-Dependence of CYP450/Substrate Incubation in Two-StepLuminescent CYP450 Reactions

Time courses were performed for incubation of CYP1A2, CYP2C8 and CYP2C9with Luc ME, CYP2C9 with H-Luc and CYP3A4 with Luc BE (FIG. 4). Lightoutput measured within 12 minutes of adding a luciferase reaction mix tothe CYP450 reaction mix increased in a linear fashion for up to 60minutes with CYP2C9 and Luc ME or H-Luc and for CYP3A4 with Luc BE. WithCYP1A2 and CYP2C8 there was a time dependent increase but the rate ofincrease declined and was increasing only modestly by 60 minutes forCYP2C8, and not at all for CYP1A2. These time courses mirror theactivity of CYP1A2, CYP2C8, CYP2C9 and CYP3A4 with the conventionalsubstrates phenacetin, paclitaxel, diclofenac and testosterone,respectively (10, 11, 12).

Example 4 Time Course of Light Input from Two-Step Luminescent CYP450Reactions

In this Example, a luminescent signal was generated after combiningluciferase reaction components with a CYP450 reaction and was monitoredover time (FIG. 5). D-luciferin derivatives were incubated in a CYP450reaction mix for 60 minutes at 37° C. before combining with a luciferasereaction mixture. In —CYP450 controls CYP450 Sf9 cell microsomes werereplaced with H₂O. Luminescence was read on a Turner Reporterluminometer beginning 3 minutes after combining the reactions and atsuccessive intervals as indicated for 284 minutes. For the CYP450s andsubstrates tested, the signals were quite stable, decaying with ahalf-life of greater than 5 hours.

Example 5 One Step CYP450/Luciferase Reaction at Room Temperature

In this Example, a procedure for a one-step CYP450/luciferase assay isprovided. Luciferin derivatives were evaluated as P450 substrates andluciferase pro-substrates using this procedure. 100 microliter CYP450reactions were prepared at pH 7.4 in an amount of KPO₄ buffer that isoptimal for a given CYP450 isoform (see two step reaction method).Reaction mixes also contained 1.3 mM NADP+, 3.3 mM glucose-6-phosphate,0.4 U/ml glucose-6-phosphate dehydrogenase, 3.7 mM MgSO₄, 0.6 mMcoenzyme A, a luciferin derivative/CYP450 substrate, 250 micromolar ATPand 21 microgram/mL thermostable luciferase from Photuris pennsylvanicaprepared as described in WO/9914336, published Mar. 25, 1999 which isincorporated by reference in its entirety). Reactions were initiated byaddition of 2.0 pmoles recombinant human CYP450 co-expressed with CYP450reductase in Sf9 cell microsomal membranes (e.g. GenTest Supersomes™)and incubation at room temperature or 37° C. Light output was measuredimmediately and continuously in a Turner Reporter or Berthold Orionluminometer.

The one-step assays were performed at room temperature (˜22° C.) withLuc ME, which was co-incubated with CYP1A2, CYP2C8 and CYP2C9 microsomepreparations, and luciferase with its requisite cofactors. Initialbaseline luminescence was measured and then light output was monitoredover time from the point reactions were initiated by adding CYP450 (FIG.6). For each reaction a time dependent increase in light output wasobserved when CYP450 was included in the reaction mix. The most robustresponse at room temperature was seen with CYP1A2. Light outputincreased to a maximum level after about forty minutes, remained steadyfor about 3 hours and then declined gradually over the remainder of theassay. Light output from CYP2C8 and CYP2C9 room temperature reactionsincreased gradually for about 3 hours, remained steady for about 1 hourthen declined gradually over the remainder of the assay. Similarone-step assays were also performed with H-Luc and CYP2C9 and with LucEE and CYP2C8 (data not shown).

Example 6 One Step CYP450/Luciferase Reaction at 37° C.

In this Example, the one step assay of Example 5 was performed at 37° C.and light was monitored over time from the point where the reactionswere initiated by adding CYP450 (FIG. 7). Cyp1A2, CYP2C8 and CYP2C9 wereassayed against Luc ME, CYP2C9 against H-Luc and CYP3A4 against Luc BE.Cyp1A2 and CYP2C9 were similar, increasing to a peak of light output byabout 30 minutes and declining thereafter. In a CYP2C8 reaction lightoutput exceeded that from a —CYP450 control but declined from initialvalues over the course of the reaction in both test and controlconditions. CYP2C9 with H-Luc was similar to Luc ME, increasing to amaximum by about 30 minutes and declining thereafter. The difference inlight output between CYP3A4 with Luc BE and a —CYP450 control wasmodest. Light output from —CYP450 controls in each case was likely aconsequence of luciferin contamination in the unreacted D-luciferinderivative preparation.

Example 7 Pooled Human Liver Microsomes in Two-Step Luminescent CYP450Reaction

A pooled human liver microsome preparation containing a mixture ofCYP450 activities was used in two-step luminescent CYP450 assays thatemployed H-Luc, Luc ME, Luc EE and Luc BE (FIG. 8) as substrates. Ascompared to Sf9 cell membranes with no CYP450 activity significantamounts of CYP450 activity was detected by the luminescent method witheach substrate. The contributions of individual isoforms to the totallight output were implied by inhibition with sulfaphenazole (CYP2C9inhibitor), alpha-naphthoflavone (CYP1A2 inhibitor) and ketoconazole(CYP2C8 and CYP3A4 inhibitor) (13). Of particular note was the nearcomplete inhibition of light output with H-Luc by the CYP2C9 selectiveinhibitor sulfaphenazole. Partial inhibition of the H-Luc reaction by100 micromolar ketoconozole is also consistent with the effect of thisinhibitor on CYP2C9 activity. This coupled with the demonstration theH-Luc is selective for CYP2C9 (FIG. 3) indicates that the microsomalactivity against H-Luc is predominantly CYP2C9. The effects ofsulfaphenazole and ketoconazole on Luc ME and Luc EE activity areconsistent with the presence of CYP2C8 activity because CYP2C8 is activeagainst both of these substrates and inhibited by both inhibitors.Inhibition of Luc BE activity by ketoconazole is consistent with thepresence of CYP3A4 and/or CYP2C8 because both isoforms are activeagainst Luc BE and both are inhibited by ketoconazole. The lack ofinhibition by alpha-naphthoflavone indicates that there is little or noCYP1A2 activity present in the microsome preparation. The slightstimulation of activity against Luc BE by alpha-naphthoflavone mayreflect the presence of CYP3A4 activity because this isoform isstimulated by alpha-naphthoflavone (16).

Example 8 Detection of CYP450 Inhibition by Known P450 Inhibitors

Luciferin derivatives as substrates for luminescent CYP40 assays shouldbe useful as probes for detecting CYP450 inhibition by drugs or otherxenobiotics. To test this hypothesis, known CYP450 inhibitors andcertain luciferase derivatives were added to the reactions and IC50swere determined. Inhibitors tested were sulfaphenazole for CYP2C9,alpha-naphthoflavone for CYP1A2, and ketoconazole for CYP2C8 and 3A4.Two-step assays were performed as described in Example 2. These drugsinhibited the reactions in a dose-dependent manner (Table 1). In manycases, the IC50s were comparable to those reported for assays with othersubstrates (13). The inhibitors were acting on the CYP450s. Inhibitionof luciferase was not detected in control assays that used luciferin assubstrate (data not shown).

Table 1 summarizes the inhibition of CYP450 reactions with conventionalsubstrates and D-luciferin derivatives by ketoconazole,alpha-naphthoflavone and sulfaphenazole. CYP450 assays with luciferinderivatives were performed in two steps essentially as described inExample 2. In this case the CYP450s were exposed to inhibitors atconcentrations ranging from about 40 nM to 10 micromolar for 10 minutesprior to exposure to a D-luciferin derivative. IC₅₀S were calculated bynon-linear regression analysis with GraphPad Prism software. Entriesmarked with an asterisk were taken from reference 13.

TABLE 1 Inhibition of CYP450 reactions by CYP450 inhibitors. Alpha-Naphptho- Sulfa- P450/substrate flavone Ketoconazole phenazoleCYP1A2/Luc ME 0.2 — — CYP1A2/ethoxyresorufin* 0.4 — —CYP1A2/phenanthrene* 3.8 — — CYP1A2/imaprine* 0.1 — — CYP2C8/Luc EE —26.4 — CYP2C8/phenanthrene* — 8.9 — CYP2C9/H-Luc — — 0.7 CYP2C9/Luc ME —— 0.4 CYP2C9/Luc EE — — 0.5 CYP2C9/diazepem* — — 0.5CYP2C9/phenanthrene* — — 0.7 CYP3A4/Luc BE — 0.06 — CYP3A4/diazepem* —0.5 — CYP3A4/phenanthrene* — 0.03 — CYP3A4/testosterone* — 0.04 — IC₅₀s(micromolar) against CYP450 isoform/substrate reactions are shown. *Fromreference 13.

Example 9 CYP450-Catalyzed Conversion of Luciferin Derivatives toLuciferin

In this Example, the conversion of Luc ME, H-Luc, Luc CEE and Luc 3PE toluciferin by cytochrome P450 enzymes was confirmed by HPLC analysis(FIG. 10). 100 micromolar Luc ME, H-Luc, Luc CEE and Luc 3PE wasincubated with CYP1A2, CYP2C9, CYP1A1 and CYP3A4, respectively inreaction volumes of 150 microliters at 37 C. At various time intervals,the reactions were stopped by the addition of Tergitol to 0.1% (v/v) andflash freezing in liquid nitrogen. 95 microliter aliquots of eachreaction mixture were subjected to fractionation by HPLC. HPLC method:High-performance liquid chromatography was performed on an HP 1050 LCsystem equipped with a multi-wavelength absorbance (HP 1050 MWD) andfluorescence detector (HP 1046A). Separation was achieved on a 5 micronAdsorbosphere HS C18 column (Alltech Associates) with a solvent gradientof 0.05M KH₂PO₄/pH 6 (solvent A) and 80:20 acetonitrile/water (solventB). The gradient conditions used were 15% B to 95% B over 10 min.Substrates were detected by absorbance at either 262 or 330 nm andLuciferin was detected by fluorescence at 520 nm (emission) with anexcitation wavelength of 330 nm. The zero time points represent theluciferin content of deoxyluciferin or luciferin 6′ methyl ether from noenzyme controls.

Example 10 Detection of CYP450 Inhibition by Known CYP450 Substrates

Luciferin derivatives as substrates for luminescent CYP450 assays wouldbe useful as probes for detecting other CYP450 substrates. Because twodistinct substrates for the same CYP450 isoform will likely compete forthe active site, it is possible to characterize known substrates and toidentify novel substrates by observing their capacity to inhibit theluminescent reactions with luciferin derivatives. To test thishypothesis, known CYP450 substrates were added to the reactions(Tassaneeyakul, W. et al (1993) “Specificity of substrate and inhibitorprobes for human cytochromes P450 1A1 and 1A2”, J. Pharmacol. Exp.Ther., 265, 401-407; Mancy, A. et al “Diclofenac and its derivatives astools for studying human cytochromes p450 active sites: particularefficiency and regioselectivity of p450 2Cs”, Biochemistry, 38,14264-14270). Substrates tested were diclofenac for CYP2C9 andphenacetin for CYP1A1 and CYP1A2. The drugs inhibited the reactions in adose-dependent manner, thus verifying the expectation that CYP450substrates can be detected by these luminescent assays (see tablebelow). IC50s were calculated by non-linear regression analysis with theprogram GraphPad PRISM™ (San Diego, Calif.). The reactions wereperformed as described in Example 2 except the first step (CYP450reaction) was in a 50 microliter reaction volume with 1 picomole ofCYP450. In the second step a 50 microliter luciferase reaction was addedto give final concentrations of 50 micrograms/mL recombinant, mutant offirefly luciferase from Photuris pennsylvanica from Promega (17), 200micromolar ATP, 0.1% tergitol (v/v), 4.0 mM MgSO₄ and 100 mM Tricine pH8.4. FIGS. 11( a)-(c) illustrate the actual inhibition curves.

TABLE 2 CYP450 isoform/substrate Diclofenac Phenacetin CYP2C9/H-Luc 13ND CYP1A1/Luc CEE ND 21 CYP1A2/Luc ME ND 25 Values shown are IC₅₀S(micromolar).

Example 11 Two-step Cyp450/Renilla Reaction and CoelenterazineDerivative Evaluation

In this Example, P450 activity was determined using coelenterazine andcoelenterazine derivatives, methoxy-coelenterazine HH and coelenterazineHH, in a two-step reaction system. In these assays, P450 acts on thecoelenterazine or coelenterazine derivatives in one of two ways. In thefirst type of reaction coelenterazine derivatives that are neithersubstrates for Renilla-type luciferases nor exhibit the characteristiccoelenterazine chemiluminescence (luminescence in the absence ofluciferase) are altered by P450 to become substrates for Renilla-typeluciferase and exhibit chemiluminescence. An example of this type ofcoelenterazine derivative is methoxy-coelenterazine HH. In the secondtype of reaction coelenterazine or coelenterazine HH, which exhibitchemiluminescence and are competent substrates for Renilla-typeluciferase, are altered by P450 resulting in a loss of chemiluminescenceand activity with Renilla-type luciferases. In both types of assay it ispossible to detect P450 activity either directly by a change inchemiluminescence or indirectly by a change in bioluminescence from aRenilla-type luciferase.

(I) Synthesis of Coelenterazine Derivatives

2-Oxo-3-phenyl-propionaldehyde

Phenylpyruvic acid (25.0 g, 152.0 mmol) was coevaporated twice with drypyridine and then redissolved in dry pyridine (250 mL). To this solutionwas added acetic anhydride (170 mL, 1.8 moles) and the solution wasstirred at ambient temperature for 15 h. The reaction progress wasmonitored by TLC. When the reaction was complete, the solution wasevaporated to a viscous syrup. The syrup was dissolved indichloromethane (700 mL) and then washed three times with 0.1 M aqueousHCl solution (3×200 mL). The organic phase was dried over anhydroussodium sulfate, filtered and evaporated to an amber-colored syrup. Thisproduct was purified by flash chromatography on silica gel (250 g) usingdichloromethane as mobile phase. Appropriate fractions were pooled andevaporated to afford 24 g of a dry solid. This material was dissolved inTHF (150 mL) and the solution was cooled in an ice-water bath. To thesolution was added dropwise oxalyl chloride (51 mL, 580 mmol). After 10min DMF (7.5 mL) was added to the reaction mixture and the reaction wasstirred for 4 h at 0° C. Toluene (100 mL) was added and the reactionmixture was evaporated to give a thick oil. This material wascoevaporated twice with toluene and the crude product was dried undervacuum for 5 h. The dried product was dissolved in a 1:1 mixture ofTHF-dichloromethane (200 mL) and the solution was cooled to −78° C. (dryice-isopropanol bath) under argon. Then, lithiumtri-tert-butoxyaluminohydride (152 mL of a 1.0 M solution in THF, 152mmol) was added at a rate such that the internal temperature of thereaction was below 60° C. After addition was complete, the reaction wasstirred below −60° C. for 10 h. The reaction was quenched by the slowaddition of 2 M aqueous HCl solution (100 mL) and the mixture wasallowed to warm to ambient temperature. The reaction mixture was dilutedwith dichloromethane (500 mL) and then washed twice with 0.1 M aqueousHCl solution (2×100 mL). The organic phase was dried over anhydroussodium sulfate, filtered and evaporated to give an amber-colored syrup.This material was purified by flash chromatography on silica gel (250 g)starting with 95:5 heptane-ethyl acetate, then with 9:1 heptane-ethylacetate as mobile phase. Appropriate fractions were pooled andevaporated to afford 13.5 g (80%) of the desired compound.

2,8-Dibenzyl-6-phenyl-7H-imidazo[1,2-a]pyrazin-3-one (Coelenterazine HH)

A solution of 2-amino-3-benzyl-5-phenylpyrazine^(20.)(2.0 g, 8.0 mmol)and 2-oxo-3-phenyl-propionaldehyde (3.0 g, 16 mmol) in ethanol (125 mL)was deoxygenated with argon gas for 20 min. To the solution was addedconcentrated hydrochloric acid (4.0 mL) and the reaction mixture washeated at reflux for 18 h. The reaction was allowed to cool to ambienttemperature and then evaporated to a brown solid. The crude product wastriturated with ethanol (40 mL) and the resulting solid material wascollected by centrifugation and then dried in a vacuum oven to afford1.28 g (41%) of the desired compound. This product was 80% pureaccording to HPLC analysis.

2,8-Dibenzyl-3-methoxy-6-phenyl-imidazo[1,2-a]pyrazine (CoelenterazineHH methyl ether)

To a stirred solution of2,8-dibenzyl-6-phenyl-7H-imidazo[1,2-a]pyrazin-3-one (0.25 g, 0.6 mmol)in dry DMF (10 mL) at ambient temperature under argon was addeddiisopropylethylamine (1.1 mL, 6.0 mmol) all at once, followed bydropwise addition of methyl iodide (0.4 mL, 6.0 mmol). After stirringfor 1 h the reaction was complete by TLC analysis. The reaction mixturewas diluted with dichloromethane (75 mL) and washed twice with water.The organic extracts were dried over anhydrous sodium sulfate, filteredand evaporated to provide a brown oil. The crude oil was purified byflash chromatography on silica gel (30 g) using dichloromethane asmobile phase. Appropriate fractions were pooled and evaporated to afford200 mg (77%) of the desired compound.

(II) P450 Assays

P450 reactions (20 microliter) containing 200 mM KPO₄ pH 7.4 (forCYP3A4) or 100 mM KPO₄ pH 7.4 for (CYP1A1, 1A2, 2B6, 2D6, 2E1) or 50 mMKPO₄ pH 7.4 (for CYP2C8, 2C19) or 25 mM KPO₄ pH 7.4 (for CYP2C9) or 100mM Tris pH 7.5 (for CYP2A6); insect cell microsomes containingbaculovirus expressed P450 (1 pmole) and P450 reductase or for no P450control reactions wild type baculovirus infected insect cell microsomes;1.3 mM NADP⁺; 3.3 mM Glucose-6-Phosphate; 3.3 mM MgCl; 0.4 units/mlGlucose-6-Phophate Dehydrogenase; and substrate (3 micromolarCoelenterazine, 3 micromolar Coelenterazine HH and 10 micromolarMethoxy-coelenterazine HH) were incubated for 60 min at 37° C.

Chemiluminescence of P450 altered coelenterazine or coelenterazinederivatives was determined immediately following addition of 80microliter of 37.5 mM Hepes pH7.4; 625 mM KCl; 0.125 mM EDTA pH 8.0;0.25% Triton X-100; 0.125% Mazu; 1.25% Glycerol; 0.25 mg/mL gelatin tothe P450 reactions. Renilla luciferase (1.2 ng/mL) (purchased fromChemicon) was added for detection of bioluminescence of P450 alteredcoelenterazine or coelenterazine derivatives.

As shown in FIG. 12, there was a large increase in bothchemiluminescence (panels C and D) and bioluminescence (panels A and B)following incubation of methyl-coelenterazine HH with CYP1A1. There weremodest increases in chemiluminescence and bioluminescence with CYP1A2,2B6 and 2C19 (2-5×). There was a significant reduction in bothchemiluminescence (panels G and H) and bioluminescence (panels E and F)following incubation of coelenterazine HH with CYP1A2 and 2E1. Finally,there was a very large reduction in both chemiluminescence (panels K andL) and bioluminescence (panels I and J) following incubation ofcoelenterazine with all of the P450 isozymes tested except 2C9 and 2A6.

Example 12 Luciferase Protection from Inhibitory Buffer by the Additionof Yeast iPPase

This Example illustrates the reversal of inhibition of aluciferase-based P450 reaction in the presence of inhibitory buffer byiPPase. As defined herein, “inhibitory” refers to a reagent (such as abuffer) that includes iPP in sufficient amounts to inhibit theluciferase reaction. A “non-inhibitory reagent” is a reagent thatincludes substantially no iPP, as measured by its effect on theluciferase reaction. The iPP content is determined empirically by thefinding that firefly luciferase is inhibited by iPP, an inhibition isrelieved by addition of iPPase and that the inhibition can be recreatedby the addition of PPi.

P450 reactions (50 microliter) contained: 1 pmole CYP1A2 (controlreactions contained SF9 membranes), 1.3 mM NADP⁺, 3.3 mMglucose-6-phosphate, 0.2 U/ml glucose-6-phosphate dehydrogenase, 3.3 mMMgCl₂, 0.01 mM Luc-ME and either 100 mM KPO₄ pH7.4 (inhibitory buffer)or 100 mM KPO₄ pH 7.4 (non-inhibitory buffer). Reactions were incubatedat 37° C. for 1 hour. The detection of luciferin generated by the P450reaction was carried out by addition of equal volume of a reagentcontaining: thermostable luciferase (100 mg/mL) from Photurispennsylvanica prepared as described in WO/9914336, published Mar. 25,1999 which is incorporated by reference in its entirety); 400 micromolarATP, 0.4% Prionex, 40 mM Tricine pH 7.8, 8 mM MgSO₄, 0.2% Tergitol.iPPase (Sigma Company, Catalog No. 11891) was added to some of thereactions. Luminescence was detected using a Berthold Orion MicroplateLuminometer.

As shown in FIG. 13 and in FIG. 15, yeast inorganic pyrophosphatase waseffective in reversing iPP inhibition of luciferase when inhibitorybuffer is used.

Example 13 Inorganic Pyrophosphatases Protect Luciferase fromPyrophosphatase Contamination

In this experiment, thermostable inorganic pyrophosphatase from threesources, New England Biolabs, Inc. (Beverly, Mass., Catalog #M0296), acommercially available yeast inorganic pyrophosphatase from Sigma (CatNo. 11891), and a pyrophosphatase isolated from Thermus thermophilus(Tth), isolated by conventional methods, were evaluated for theirefficiencies at reversing the effect of iPP contaminated buffer in aluciferase-based P450 reaction.

In this experiment, P450 reaction mixtures (50 microliter) wereprepared. The reactions contained: 1 pmole CYP1A2 (control reactionscontained Sf9 membranes), 1.3 mM NADP⁺, 3.3 mM glucose-6-phosphate, 0.2U/mL glucose-6-phosphate dehydrogenase, 3.3 mM MgCl₂, 0.01 mM LucME, andeither 100 mM KPO₄ pH7.4 (inhibitory buffer) or 100 mM KPO₄ pH 7.4(non-inhibitory buffer. Reactions were incubated at 37° C. for one hour.The detection of luciferin generated by the P450 reaction was carriedout by addition of equal volume of a reagent containing 100 mg/mLthermostable Luciferase from Photuris pennsylvanica prepared asdescribed in WO/9914336, published Mar. 25, 1999 which is incorporatedby reference in its entirety), 400 mM ATP, 0.6% Prionex, 40 mM TricinepH 7.8, 8 mM MgSO₄, 0.2% Tergitol, 0.02% Mazu. iPPase from New EnglandBiologics, Inc. (Beverly, Mass., Catalog #M0296), Sigma (Cat No. 11891),or Thermus thermophilus (Tth), isolated by conventional methods wasadded to some of the reactions. Following incubation at room temperaturefor 30 minutes luminescence was detected using a Berthold OrionMicroplate Luminometer.

As shown in FIG. 14, inorganic pyrophosphatase from different sourcesreversed inhibition of luciferase when inhibitory KPO₄ buffer is used ina P450 reactions. Reaction conditions, temperature and enzymeconcentrations were found to affect the efficiencies of the variousiPPase enzymes in reversing iPP inhibition (data not shown).

Example 14 Protection of Luciferase from Added iPP Using Yeast iPPase

This Example illustrates iPPase reversal of inhibition of a luciferasereaction in the presence of added iPP and shows that the performance of200 mM non-inhibitory KPO₄ Buffer with 3 mM NaPPi is similar to that of200 mM inhibitory KPO₄ buffer and that iPPase reverses the effect of theadded NaPPi.

The reactions contained: 100 mM Tricine pH 8.4, 10 mM MgSO₄, 0.1%Tergitol, 0.01% Mazu, 50 mg/mL thermostable luciferase (from Photurispennsylvanica prepared as described in WO/9914336, published Mar. 25,1999 which is incorporated by reference in its entirety), 200 mM ATP,0.2% Prionex, 0.5 mM luciferin. All reactions contained either 200 mMnon-inhibitory KPO₄ pH 7.4 or 200 mM inhibitory KPO₄ pH7.4. iPPase(Sigma 11891) was added to some of the reactions to a finalconcentration of 2 units/mL. Sodium pyrophosphate (NaPPi) was added tosome reactions to a final concentration of 3 mM. The reactions wereperformed at room temperature. Luminescence was detected using aBerthold Orion Microplate Luminometer.

As shown in FIG. 15 inorganic pyrophosphatase was effective in reversinginhibition of luciferase when inhibitory KPO₄ buffer is used. Withoutbeing bound to a mechanism, the inventors observed that the addition ofiPP to an otherwise active buffer can recreate an inhibitory buffer, andthat the inhibition can be reversed by the addition of iPPase. Thesefindings imply that the inhibitor is iPP.

Example 15 Cell-Based Luminescent CYP450 Assay

In this Example, a cell-based luminescent CYP450 assay is described.Inducers of CYP450 gene expression were evaluated for their effect onCYP450 activity. Primary hepatocytes from sexually mature male SpragueDawley rats were obtained cryopreserved from Xenotech, LLC (Kansas City,Kans.). On the first day cells were thawed as recommended by thesupplier and the percentage of live cells estimated by the method oftrypan blue exclusion. Approximately 1.5×10⁵ cells per cm² were seededon collagen-coated 24-well tissue culture plates. Cells were cultured at37° C., 95% relative humidity and 5% CO₂ in 0.3 mL/well HepatoZyme SFMmedium supplemented with 2 mM L-glutamine and 1× penicillin-streptomycin(Life Technologies, Inc., Rockville, Md.). Initially cells were allowedto attach to plates for 6 hours then the medium was replaced with freshmedium supplemented to 0.25 mg/ml with Matrigel™ (BD Biosciences,Bedford, Mass.). Medium was changed daily.

On the third day after seeding of cells, culture medium was removed andreplaced with 0.3 ml medium containing CYP450 gene inducers or theirvehicle controls. On the fourth day medium was removed and replaced withfresh induction or vehicle control medium so that cells were exposed for2 days.

On the fifth day induction and vehicle control media were replaced withfresh medium that contained luminogenic CYP450 substrates. At the end ofthe incubation period with luminogenic substrate, two types ofluminescent assays were performed. Both assay formats were possiblebecause the luminogenic substrates enter cells, likely by passivediffusion. The first type of assay is possible because the luciferinproduct of CYP450 reaction exits cells, again likely by passivediffusion. For the first type of assay a sample of medium was removedand combined with an equal volume of a luciferin detection reagent (200mM tricine, pH 8.4, 100 micrograms/mL thermostable mutant of fireflyluciferase from Photuris pennsylvanica (Ultra Glow™ luciferase,available from Promega, Corp.), 400 micromolar ATP, 20 mM MgSO₄ and 2%Tergitol) to initiate a luminescent reaction. For blank determinationsfor the first assay type luminogenic substrate was withheld from somewells but combined with an aliquot of medium after it was first combinedwith the luciferin detection reagent. For the second type of assay, anequal volume of luciferin detection reagent was added directly to thecell culture medium to stop CYP450 activity, produce a cell lysate andinitiate a luminescent reaction. For blank determinations for the secondreaction type luminogenic substrate was withheld from some wells butadded to these wells after the luciferin detection reagent was firstadded. Aliquots of both types of reactions were transferred to white,opaque 96-well plates and luminescence was read on a Fluostar Optimaluminometer (BMG, Inc.). Luminescence values from blank wells weresubtracted from the values of corresponding wells. The results are shownin FIG. 16.

Example 16 Stabilization of Luminescent Signal Using LuciferaseInhibitors

In this Example, two luciferase competitive inhibitors,2-amino-6-methylbenzothiazole (AMBT) or2-(4-aminophenyl)-6-methylbenzothiazole (APMBT), were evaluated todetermine their effect on stabilizing a luminescent signal.

50 microliter CYP1A1 reactions (0.5 μmol recombinant CYP1A1 enzyme, 30μM Luciferin chloroethyl ether, 100 mM KPO₄, 1.3 mM NADP⁺, 3.3 mMglucose-6-phosphate, 3.3 mM MgCl₂, 0.02 unit glucose-6-phosphatedehydrogenase) were incubated at 37° C. for 20 min. After theincubation, 50 microliters of a luciferin detection reagent (100micrograms/mL thermostable luciferase (from Photuris pennsylvanica), 400micromolar ATP, 0.6% Prionex, 2 units/mL iPPase, 200 mM Tricine pH 8.4,20 mM MgSO₄, 2% Tergitol) containing either 100 micromolar APMBT, 100micromolar AMBT, or no inhibitor were added to each CYP1A1 reaction.Luminescence was read immediately and at subsequent 5 minute intervalsfor 1 hour. The results are shown in FIG. 17.

As shown in FIG. 17, inhibition of luciferase by an inhibitor such as2-(4-aminophenyl)-6-methylbenzothiazole (APMBT) or 2-amino-6-methylbenzothiazole (AMBT) stabilizes the luminescent signal in a luminescentCYP450 assay.

While the present invention has now been described and exemplified withsome specificity, those skilled in the art will appreciate the variousmodifications, including variations, additions and omissions, that maybe made in what has been disclosed herein without departing from thespirit of the invention. Accordingly, it is intended that thesemodifications also be encompassed by the present invention and that thescope of the present invention be limited solely by the broadestinterpretation that lawfully can be accorded the appended claims.

REFERENCES

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What is claimed is:
 1. The compound having the structure

or a salt thereof.
 2. A composition comprising a compound of claim 1 andan aqueous carrier.
 3. The composition of claim 2, further comprising apyrophosphatase.
 4. The composition of claim 3 wherein thepyrophosphatase is an inorganic pyrophosphatase.